Medane genes and proteins

ABSTRACT

This invention relates to a novel DNA sequence encoding a bHLH transcription factor in vertebrates, preferably mammals, e.g. in mice or humans, as well as the expressed transcription factor. The invention further relates to vectors containing said DNA sequences and host cells transformed by these vectors. Furthermore, the invention encompasses antibodies specific for the transcription factors as well as the use of the DNA sequences and transcription factors in the diagnosis or therapy of neurodegenerative diseases, e.g. Parkinson&#39;s disease. The present invention further relates to an ex vivo method of producing dopaminergic cells and the therapeutic use of these dopaminergic cells.

RELATED APPLICATIONS

[0001] This application is a continuation of PCT patent applicationnumber PCT/EP02/01077, filed Feb. 1, 2002, which claims priority toGerman patent application number 10104584.0, filed Feb. 1, 2001, thedisclosures of each of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

[0002] This invention relates to a novel DNA sequence encoding a bHLHtranscription factor in vertebrates, preferably mammals, e.g. in mice orhumans, as well as the expressed transcription factor. The inventionfurther relates to vectors containing said DNA sequences and host cellstransformed by these vectors. Furthermore, the invention encompassesantibodies specific for the transcription factors as well as the use ofthe DNA sequences and transcription factors in the diagnosis or therapyof neurodegenerative diseases, e.g. Parkinson's disease. The presentinvention further relates to an ex vivo method of producing dopaminergiccells and the therapeutic use of these dopaminergic cells.

BACKGROUND ART

[0003] Up to now, a variety of DNA sequences has been identified, whichcode for vertebrate bHLH transcription factors. For example,approximately 140 human bHLH transcription factors have been identifiedbased on the human genome sequences. However, the knowledge is limitedto the sequence data per se and no function of these transcriptionfactors has been elucidated.

[0004] Neurotransmitters are endogenous substances that are releasedfrom neurons and produce a functional change in the properties of thetarget cells. The amino acid tyrosine is the precursor for threedifferent amine neurotransmitters that contain a chemical structurecalled a catechol. These neurotransmitters are collectively calledcatecholamines and include dopamine, norepinephrine, and epinephrine.The catecholamine neurotransmitters contain the enzyme tyrosinehydroxylase (TH), which catalyses the initial steps in catecholaminebiosynthesis (Nagatsu et al., 1964) the conversion of the amino acidL-tyrosine into a compound called L-dopa (3,4-dihydroxyphenylalanine).

[0005] The Catecholaminergic system is one of the major mono-aminergicsystems in the brain stem. Catecholamines neurons have been shown to bein regions of the nervous system involved in the regulation of movement,mood, attention, and visceral function and are composed of dopamine,noradrenaline and adrenaline producing neurons (reviewed in Smeets andReiner, 1994).

[0006] The dopaminergic system is highly organised topographically. Inmammals, the dopaminergic neurons (DA) reside in the telencephalon,diencephalon and midbrain. DA neurons of the adult mammal have beenplaced into nine distinct groups (Specht et al., 1981; Bjorklund andLindvail, 1984; Voorn et al., 1988). The telencephalon contains twosmaller groups of DA neurons restricted to the olfactory bulb (group 16)and the retina (group A17).

[0007] The most prominent groups are the so-called mesencephalicdopaminergic neurons (mesDA) residing in the ventral midbrain (groupsA8, A9, A10) and in the diencephalon groups A11-A15 involved in therelease of pituitary hormones.

[0008] The mesDA are a limited set of neurons and can be subdivided intothree groups, the ventral tegmental area innervate the neocortex (groupA10), the substantia nigra pars compacta innervate the striatum (groupA9) and the retrorubral nucleus (group A8). In the mouse, the generationof these DA cells can be monitored from approximately embryonic day 11.5(E11.5) by expression of TH.

[0009] The mammalian DA neurons regulate behaviour and voluntarymovement control, reward-associated behaviour, and hormonal homeostasisand has been implicated in psychiatric and affective disorders (Grace etal., 1997). The selective degeneration of dopaminergic neurons withinthe mesDA system is the direct cause of the motor disordercharacteristic of Parkinson's disease (Jellinger, 1973; Forno, 1992;Golbe, 1993). Whereas overstimulation of ventral tegmental DA neuronshas been implicated in affective disorders like manic depression andschizophrenia (Ritz et al., 1987) and in behavioural reinforcement anddrug addiction (Seeman et al., 1993).

[0010] Generation of cellular diversity in the developing mammalianbrain involves cascades of secreted signalling molecules that acts inthe specification of the distinct neuronal cell types. Thiscoarse-grained pattern is subsequently reinforced and refined bydiverse, locally acting mechanisms resulting in a precise regionalvariation in cell identity (Lumsden et al., 1996). The network by whichmesDA neurons assume their specific identity and are confined to theventral part of the midbrain in mouse embryos is poorly understood. Theprogenitors for this neuronal cell type lie on the ventral part of themidbrain as early as E9 and they differentiate in this region at a timebetween E9 and E14 (Hynes and Rosenthal, 1999). Their specification isdependent on multiple co-operating epigenetic signals like the presenceof ventrally expressed Sonic Hedgehog (Shh), a secreted proteinimportant for ventral cell fates in the central nervous system (CNS)(Echelard et al., 1993; Ericson et al., 1995). Another secreted proteinfrom the mid-/hindbrain (MHB) boundary important for the dopaminergicphenotype is Fibroblast growth factor-8 (FGF8). A combined signalling ofthese two secreted proteins has been shown to mediate the generation ofdopamine progenitors cells (Ye et al., 1998), but finally they arespecified in response to yet unidentified inductive intracellularsignal. Although some of these intracellular signals, the homeobox genePtx3 (Smidt et al., 1997) and the orphan nuclear hormone receptor Nurrl(Castillo et al., 1999), have been shown linked to the TH pathway, noneof these early signals explain the process underlying the specificationof DA neurons, (Hynes and Rosenthal 1999).

[0011] Thus, the intracellular transcription factor required specifyinginduction of midbrain-dopamine cell lineage remained to be identified.

[0012] The understanding of the basis mechanisms of vertebrate celldifferentiation has been greatly advanced by the findings oftranscription factors such as the basic helix-loop-helix (bHLH) (Lee,1997). The bHLH proteins comprise evolutionarily ancient transcriptionfactors united by conservation solely within the bHLH domain (Murre etal., 1994). bHLH proteins have been found to function as transcriptionalregulators in a variety of developmental processes (Olson, 1990, Cabreraand Alonso 1991, Van Doren et al., 1992, Martinez et al., 1993),regulating the determination of neural progenitor cells (Campos-Ortega,1993) and other cell fate decisions (Carmena et al., 1995, Corbin etal., 1991, Ruohola et al., 1991, Xu et al., 1992).

[0013] A special class of bHLH proteins is defined by the translationalproducts of the Drosophila genes hairy (Rushlow et al., 1989) and the E(spl) (Kläambt et al., 1989; Knust et al., 1992; Delidakis andArtavanis-Tsakonas, 1992). In Drosophila, hairy-related bHLH factors areinvolved in delimiting expression territories and/or domains of cellspecification within the embryo and larva, controlling cell fatespecification choices during multiple developmental processes, includingneurogenesis and myogenesis, where E (spl) factors includes 7 clusteredsmall bHLH proteins comprising the majority of direct transcriptionaltargets of Delta/Notch signalling (Fischer and Caudy, 1998).

[0014] During development, many cell type specifications in higheranimals are controlled by intercellular communication governed by theNotch signalling pathway, a gene that controls cellular differentiation,establishment of sharp boundaries of gene expression and generation ofcell-type diversity (Artavanis-Tsakonas et al., 1995). In Drosophila,Notch target genes include the E (Spl) genes (Jennings et al., 1994; deCelis et al., 1996) and the Notch signalling pathway is shown to beconserved in mammalian neurogenesis (de la Pompa et al., 1997),including HES-1 gene (Jarriault et al., 1995).

[0015] There have been suggestions that also Mammalian homologues ofDrosophila bHLH are playing an important role as regulators of cell fatedecisions in the developing nervous system and inducers of neuronaldifferentiation at the level of gene transcription. (Reviewed by Jan andJan, 1993; Lee, 1997) However, no specific function of such a bHLHtranscription factor in the developing nervous system has beenidentified yet.

SUMMARY OF THE INVENTION

[0016] Therefore, it is the object of the present invention to providenew bHLH transcription factors, which are involved in and can be usedfor the specification of cells in the developing nervous system, inparticular in the induction of dopaminergic neurons.

[0017] Herein disclosed are the vertebrate Medane genes, which are novelbasic-helix-loop-helix (bHLH) genes involved in the specification anddifferentiation of neural cells, in particular in the induction ofdopaminergic neurons.

[0018] The DNA sequences of the invention encode bHLH transcriptionfactors which show some sequence homology to the previously describedhairy and Enhancer of split [E (spl)] genes of Drosophila. Therefore,this DNA sequence is generally termed Medane (for MesencephalicDopaminergic neurons E (spl) and hairy related gene).

[0019] Surprisingly, it turned out that Medane was capable ofspecifically inducing neurotransmitter secreting cells in vertebrates.Unexpectedly, the inventors found out that the development ofdopaminergic neurons could be induced by ectopic expression of Medane invertebrates.

[0020] Therefore, this invention preferably finds application for thesubstitution of degenerated or lost dopaminergic neurons in vertebrates.Thus, the present invention provides a new therapy, by which thedrawbacks of the prior art therapies, i.e. the transplantation ofembryonic mesencephalic cells, can be avoided. Following the prior arttransplantations, these cells showed a poor survival rate and dopamineproduction in the treated patients.

[0021] As used herein, the term specification or determination means thecommitment of a cell to a particular path of differentiation, eventhough there may be no morphological features that reveal thisdetermination (is not yet expressing the characteristic phenotype). Theterm differentiation means a process in the development of amulticellular organism by which cells become specialized for particularfunction.

[0022] The DNA sequences of mouse and human Medane genes according tothe present invention are disclosed in Seq. ID. No. 3 and 4 for thegenomic DNA sequence and in Seq. ID. No. 1 and 2 for the cDNA sequence.

[0023] This invention is directed to said Medane genes, fragmentsthereof and the related cDNA which are useful, for example, asfollows: 1) to produce transcription factors by biochemical engineering;2) to prepare nucleic acid probes to test for the presence of the Medanegene in cells of a subject: 3) to prepare appropriate polymerase chainreaction (PCR) primers for use, for example, in PCR-based assays or toproduce nucleic acid probes; 4) to identify Medane transcription factorsas well as homologues or near homologues thereto; 5) to identify variousmRNAs transcribed from Medane genes in various tissues and cell lines,preferably human; and 6) to identify mutations in Medane genes.

[0024] The invention further concerns the hitherto unknown mammaliantranscription factors, encoded by the Medane gene.

[0025] The Medane gene encodes a protein of 241 amino acids andfunctions in the nucleus as determined by cytogenetic studies. Medane isspecifically expressed in the precursors of dopaminergic neurons and itsexpression starting, for example, at mouse embryonic day 9 (E9), isconfined to the ventral part of the developing mouse midbrain, wheremesencephalic dopaminergic neurons (mesDA) appear later on. In situhybridisation studies show a spatio-temporal correlation between Medaneand Tyrosine hydroxilase (TH) expression along mouse catecholaminergicneurons development, and show that Medane is expressed in the precursorcells that will give rise to this neuronal cell lineage. Moreover,ectopic expression of Medane in vivo by electroporation of zebrafish(Danio rerio) embryos shows specification and differentiation of newclusters of TH positive cells. Taken together, these results indicatethat Medane is a unique bHLH and the earliest transcription factormarking the mesDA neurons, and is involved in developmentaldetermination and early commitment of mesDA neuronal lineage.

[0026] It will be understood that the practice of the invention is notlimited to the use of the exact DNA sequence as defined in Seq. ID. No.1-4. Modifications to the sequences, such as deletions, insertions, orsubstitutions in the sequence which produce silent changes in theresulting protein molecule are also contemplated.

[0027] For example, alterations in the gene sequence which result in theproduction of a chemically equivalent amino acid at a given site arecontemplated. Preferably, amino acid substitutions are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, i.e., conservative amino acidreplacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are typically in the range ofabout 1 to 5 amino acids. The variation allowed may be experimentallydetermined by systematically making insertions, deletions, orsubstitutions of amino acids in a polypeptide molecule using recombinantDNA techniques and assaying the resulting recombinant variants foractivity.

[0028] Nucleotide changes which result in alteration of the N-terminaland C-terminal portions of the protein molecule frequently do not alterprotein activity, as these regions are usually not involved inbiological activity. It may also be desirable to eliminate one or moreof the cysteines present in the sequence, as the presence of cysteinesmay result in the undesirable formation of multimers when the protein isproduced recombinantly, thereby complicating the purification andcrystallization processes. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

[0029] Therefore, where the phrase “DNA sequence” is used in either thespecification or the claims, it will be understood to encompass all suchmodifications and variations which result in the production of abiologically equivalent Medane protein, i.e. a bHLH transcriptionfactor. In particular, the invention contemplates those DNA sequenceswhich are sufficiently duplicative of the sequences disclosed so as topermit hybridization therewith under standard high stringency southernhybridization conditions, such as those described in Maniatis et al.(Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory,1982).

[0030] Furthermore, said variants also comprise nucleic acid changes dueto the degeneracy of the genetic code, which code for the same orfunctionally equivalent transcription factor as the nucleic acidsspecifically defined herein.

[0031] According to a further aspect, the present invention is directedto a purified isolated mouse/human transcription factor for theinduction of dopaminergic neurons comprising the amino acid of Seq. ID.No. 5/6 and homologues or fragments thereof which retain biologicalactivity.

[0032] Short specific protein domains can be used to internalizeproteins with a specific function into live cells across the blood-brainbarrier (1-6). This signal peptide sequence is necessary and sufficientfor nuclear internalisation, and can be used as a importing vehicle forcellular import of exogenous proteins when fused to them (7-8).

[0033] Therefore, the present invention is also directed to fusionproteins, comprising the herein described transcription factor as wellas a signal peptide, which allows the delivery of said transcriptionfactor into a target cell. For example, the TAT sequence can be usedfere, which triggers the internalization of genetically fused proteinsinto the nucleus in a high efficiency manner (2-4; 11). It was reportedthat TAT-beta-galactosidase fusion protein was transduced rapidly intocells, reaching near maximum intracellular concentrations in less than15 min (4).

[0034] Therefore, the present invention also provides for a noninvasiveintracellular way to deliver the functional properties of Mdn proteininto target cells and/or tissues, therefore mediating cell fatedecisions, according to the functional properties of the Mdn proteindescribed in this patent.

[0035] A Tat sequence can be added at the N- or C-terminus of the Mdnprotein to mediate out-side-in protein importation. A Histidine taqsequence (His×6) can also be introduced to purify the protein. Anepitope tag could also be included in order to detect and/or follow theimported fusion protein in targeted cells using a specific antiepitopeantibody. The recombinant protein might then e.g. be expressed in aninsect cell line, mediated by the baculovirus expression system (Lifetechnologies), then purified and tested for biological properties.

[0036] In vivo and/or in vitro protein transduction of such a biologicalactive fusion protein (Mdn-signal peptide) results in delivery of thebiologically active Mdn-protein properties in tissue or cells. Thetranslocation of such bioactive Mdn fusion-protein into the nucleus ofthe targeted cells or tissue, can influence nuclear activity which couldinduce an appropriate transcriptional response in order to activatesignal transduction pathways to conduct Dopaminergic-cell fatespecification and differentiation, conducted by the functionalproperties of Mdn protein described herein. To achieve thisphysiological implications, Mdn-signal peptide fusion-protein can bedelivered either in vitro (in preparation for tissue or celltransplantation) or in vivo (for specific neuronal identityregeneration) mediated by injection into the lateral ventricles of thecentral nervous system of the patient, or by intraperitoneal injection.In post of the intraperitoneal claim, recent studies in mice supportsthe idea that intraperitoneal injection of large proteins fused to theprotein transduction domain of the human immunodeficiency virus TATprotein results in delivery of the biologically active fusion protein toall tissues in mice, including the brain (12).

[0037] Taken into account altogether, the invention described hereallows direct internalization of exogenous Mdn-signal peptide fusionprotein by intact live cells into living organism (patients) or intocultured cells, in bulk concentration, in the context of proteintherapy, as well as for functional studies with model organisms, giventhe functional properties of Mdn protein, the low toxicity of this typeof protein delivery and the high efficiency of internalization by all ofthe cells.

[0038] The invention further relates to the biochemical engineering ofthe Medane genes, fragments thereof or related cDNA.

[0039] For example, said gene or a fragment thereof or related cDNA canbe inserted into a suitable expression vector. The host cells can betransformed with such an expression vector and an Medane transcriptionfactor is expressed therein. Such a recombinant protein or polypeptidecan be glycosylated or nonglycosylated, preferably glycosylated, and canbe purified to substantial purity. However, it is possible to produceproteins which are synthetically or otherwise biologically prepared.

[0040] Numerous vectors suitable for use in transforming bacterial cellsare well known. For example, plasmids and bacteriophages, such as lambdaphage, are the most commonly used vectors for bacterial hosts, and forE. coli in particular. In both mammalian and insect cells, virus vectorsare frequently used to obtain expression of exogenous DNA. In particularmammalian cells are commonly transformed with SV40 or polyoma virus; andinsect cells in culture may be transformed with baculovirus expressionvectors. Yeast vector systems include yeast centromere plasmids, yeastepisomal plasmids and yeast integrating plasmids.

[0041] Alternatively, the transformation of the host cells can beachieved directly by naked DNA without the use of a vector. Productionof Medane by either eukaryotic cells or prokaryotic cells iscontemplated by the present invention. Examples of suitable eukaryoticcells include vertebrate cells, plant cells, yeast cells and insectcells. Preferably, mammalian stem cells are used. A far as human cellsare concerned, embryonic and adult stem cells are preferred. Suitableprokaryotic hosts, in addition to E. coli, include Bacillus subtilis.

[0042] The invention also relates to a method for producing atranscription factor/polypeptide comprising growing a culture of thecells of the invention in a suitable culture medium, and purifying theprotein from the culture.

[0043] The bHLH transcription factors of this invention areserologically active, immunogenic and/or antigenic. They can further beused as immunogens to produce specific antibodies, polyclonal and/ormonoclonal.

[0044] These specific antibodies can be useddiagnostically/prognostically and may be used therapeutically. Medanespecific antibodies can be used, for example, in laboratory diagnostics,using immunofluorescence microscopy or immunohistochemical staining, asa component in immunoassays for detecting and/or quantitating Medaneantigen in, for example, clinical samples, as probes for immunoblottingto detect Medane antigen, in immunoelectron microscopy with colloid goldbeads for localization of Medane proteins/polypeptides in cells, and ingenetic engineering for cloning the Medane gene or fragments thereof, orrelated cDNA. Such specific antibodies can be used as components ofdiagnostic/prognostic kits, for example, for in vitro use onhistological sections. Still further, such antibodies can be used toaffinity purify Medane proteins and polypeptides.

[0045] The invention further relates to a composition comprising ahybridoma which produces a monoclonal antibody having bindingspecificity to any one of the disclosed transcription factors.

[0046] Antibodies are normally synthesized by lymphoid cells derivedfrom B lymphocytes of bone marrow cells. Lymphocytes derived from thesame clone produce immunoglobulin of a single amino acid sequence.Lymphocytes cannot be directly cultured over long periods of time toproduce substantial amounts of their specific antibody. However, Kohleret al., 1975, Nature, 256:495, demonstrated that a process of somaticcell fusion, specifically between a lymphocyte and a myeloma cell, couldyield hybrid cells (“hybridomas”) which grow in culture and produce aspecific antibody called a “monoclonal antibody”. Myeloma cells arelymphocyte tumour cells which, depending upon the cell strain,frequently produce an antibody themselves, although “non-producing”strains are known.

[0047] The invention further relates to a recombinant non-humanmammalian in which the DNA sequence of claim 1 has been inactivated.Preferably, a recombinant mouse is provided, in which the DNA sequenceof Seq. ID. No. 5 has been inactivated. Thus, an animal model may beestablished using such a recombinant knock-out mouse. These animalmodels allow further insights in the aetiology of several disorders inconnection with degeneration of neural cells.

[0048] The invention still further concerns nucleic acid probes that aresubstantially complementary to nucleic acid sequences of the Medanegenes. Preferred nucleic acid probes of this invention are those withsequences substantially complementary to the sequences of claims 1-9.The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or doublestranded nucleic acids. They maybe labelled by nick translation, Klenow filling reaction, PCR or othermethods well known in the art. The preparation and/or labelling of theprobes presented in the invention is described in Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

[0049] Test kits of this invention can comprise such probes which areuseful diagnostically/prognostically for neurodegenerative diseases.Preferred test kits comprise means for detecting or measuring thehybridisation of said probes to the Medane gene or to the mRNA productof the Medane gene, such as a visualizing means.

[0050] Immunoassays can be embodied in test kits which comprise Medaneproteins/polypeptides and/or Medane-specific antibodies. Such test kitscan be in solid phase formats, but are not limited thereto, and can alsobe in liquid phase format, and can be based on ELISAS, particle assays,radiometric or fluorometric assays either unamplified or amplified,using, for example, avidin/biotin technology.

[0051] As such, the Medane transcription factors are useful both in vivoand in vitro, in growth, maintenance and regeneration of nerve cells ofthe central nervous system, especially in dopaminergic precursor cells.

[0052] According to a preferred emodiment, the present inventioncomprises an ex vivo method of producing dopaminergic neurons, whichcomprises the following steps: providing neural embryonic stem cells,neural adult stem cells and/or embryonic stem cells; contacting saidcells with an effective amount of the transcription factor of thepresent invention; culturing said cells under conditions, which allowthe specification and differentiation to dopaminergic neurons; andrecovering the mature dopaminergic neurons. Furthermore, the presentinvention encompasses dopaminergic neurons, which are obtainable by thisex vivo method. These neurons may also be present in a composition,which comprises an effective amount of the dopaminergic neurons incombination with a pharmaceutically acceptable carrier.

[0053] In view of the evident role in differentiation, the Medaneprotein can also be used as a regeneration factor. In particular, Medanemay be useful in the treatment of neurodegenerative diseases, forexample Parkinson's disease. However, the term “neurodegenerativediseases” as used herein also encompasses all other diseases in whichthe dopaminergic system is involved, p.e. affective disorders like manicdepression and schizophrenia and in behavioural reinforcement and drugaddiction.

[0054] The Medane DNA gene and protein is useful in the treatment ofprogenitor cells, e.g. stem cells, to promote the differentiation ofthese cells to mature neural cells, in particular dopaminergic neuralcells. In general, human embryonic stem cells are useful for the ex vivoculturing of neural cells, which then are administered to a patientsuffering from a neurodegenerative disease.

[0055] Alternatively, adult stem cells isolated from the patient to betreated, may be differentiated ex vivo to fully developed neural cellsand then returned to the patient for the substitution of degenerated orlost neural cells.

[0056] Thus, an in vivo administration of Medane is significantlysimplified by the discovery of the gene sequence, particularly intreatment of central nervous system injury.

[0057] The identification of the gene and its sequence permitsconstruction of transgenic cells such as fibroblasts, monocytes, ormacrophages, which may be engineered to permit expression of the Medanegene and used as an implant for treatment of neurodegenerativedisorders, or any conditions in which enhancement of nerve cell growthand/or regeneration would be desirable.

[0058] Moreover, the therapeutic use of the Medane transcription factoris not limited to treatment of humans alone. In fact, in view of theconserved nature of this protein among distantly related species,administration of Medane in any form may be beneficial for veterinaryapplication as well. Therapeutic compositions comprise Medane in anamount effective to induce the desired biological activity incombination with a pharmaceutically acceptable liquid or solid carrier.Alternately, the composition may comprise a pharmaceutically acceptableaggregation of compatible transgenic cells capable of expressing Medanein vitro, as an implant for central nervous system regeneration ordifferentiation treatment.

[0059] As used herein, the abbreviation “MDN/MDN” is related to humanDNA and amino acid sequences, respectively; the abbreviation “Mdn/Mdn”to mouse DNA and amino acid sequences, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060]FIG. 1 Genomic organisation of Mdn/MDN genes. The exons andintrons are numbered and drawn to scale. Open boxes are noncodingregions; black boxes represent the translated region. The bHLH domainand the Orange domain are depicted, as well as the bipartite nucleartranslocation signal and proline rich region.

[0061]FIG. 2 Alignment of bHLH domains of hairy/E (spl)-related genesand Mdn. Alignment was generated using the Vector NTI Package programs.Bold letters depicts amino acid residues conserved among at least fourbHLH proteins. Grey letters depicts similar amino acid residues. A dashindicates spacing between amino acids to achieve best alignment.Accession numbers for: Hesl (gi 475014); Hes3 (gi 7594823); Her6 (gi1279398); Her3 (gi 1279394); Her8b (gi 10863869); hes2 (gi 6680207);hes3 (gi 6680209); Hes4 (gi 11423215); hes5 (gi 6754182); SHARP1 (gi2267587); E (spl) m7 (gi 85074); hairy (gi 85137); DPN (gi 3913501);DEC1 (gi 11414986); HEY1 (gi 7018332); Her1 (gi 10880827); Her2 (gi1279392); Her4 (gi 1279396); her7 (gi 7576909); her5 (X95301).

[0062]FIG. 3

[0063] Subcellular localisation of Mdn. Fluorescent image of transfectedhuman embryonic kidney 293 cells with 0.5 and 2 μg of EGFP-N1 blankvector (a+b, respectively). 0.5 and 2 μg Mdn-EGFP-N1 fusion protein(c+d, respectively). The same cells as above visualized with fluorescentlight combined with phase-contrast (e). A dashed white line isdelimiting the periphery of the cell.

[0064]FIG. 4

[0065] Tissue distribution of Mdn/MND transcripts. a) The Mdn mRNA wasdetected only in testis tissue when an 844 bp partial cDNA was used toprobe a mouse adult northern blot. b) Mouse foetal Northern blot showingstarting expression of Mdn as early as E8.75. We note a pick ofexpression at E11 in the ventral part of the mesencephalon. Longerexposures did not reveal other bands. c) The MDN mRNA was not detectedin any tested tissue. Equal loading of mRNAs was checked as shown in thefigure.

[0066]FIG. 5

[0067] Whole mount in situ hybridisation of Mdn. Mouse E9.5 embryoshowing the pattern of expression of Mdn restricted to the developingventral mesencephalon anterior to the isthmic organiser.

[0068]FIG. 6

[0069] Expression of Medane and TH at in horizontal sections of E12.5embryos. At E12.5 Medane is strongly expressed in specific regions ofthe ventricular zone. It is most prominent in the dorsal part of themesencephalic vesicle, the substantia nigra (sn; 1, 2), the hypothalamus(hyp; 3, 4), locus coeruleus (LC, 4, 5), in the ganglionic eminences,the future striatum (str; 5, 6) and also in the spinal cord (sc; 6).Parallel sections hybridised with a probe recognizing TH (1″-6″)revealed, that TH is expressed in regions close to Medane expressingdomains.

[0070] TH-expressing cells are located further away from the ventricularzone, than Medane expressing cells. This expression pattern suggeststhat Medane might be expressed in cells in the ventricular zone destinedto become TH-expressing cells. Also the stronger expression of Medane inthe substantia nigra as compared to the still relative weak expressionof TH suggests that Medane might be expressed before TH in this region.An additional expression domain of TH, which does not show Medaneexpression at this time point is the ganglion tf the vth cranial nerve.

[0071]FIG. 7

[0072] Expression of Medane and TH in coronal sections of E14.5 embryos.At E14.5 Medane is still strongly expressed in the ventricular zone ofthe striatum (str), discrete points in the ventricular zone of thethalamus (th) and the hypothalamus (hyp), the zone inserta (zi) and thedorsal part of the mesencephalic vesicle, representing the superior andinferior colliculus (SC, IC, 1-12). Compared to the TH-expression it isnot expressed in the olfactory bulb (ob) and not any more in thesubstantia nigra, the locus coerulus, and caudal noradrenergic groups(na, 1″-12″). This expression pattern is consistent with the idea, thatMedane expression precedes TH-expression in cells destined to becomecatecholaminergic cells.

[0073]FIG. 8

[0074] Expression of Medane in a mid sagital section of an E16.5 embryo.Expression of Medane is now restricted to the ventricular andsubventricular zone of the striatum (str), a region where the neuronsmigrating to the olfactory bulb (ob) are located. Indeed, Medaneexpressing cells can be found along the so called rostral migratorystream just until there entrance into the olfactory bulb. Again thisfits the idea, that cells destined to become TH-positive cells expressMedane before they express TH (cx=cortex, hc=hippocampus).

[0075]FIG. 9

[0076] Expression of Medane in coronal sections of an E18.5 embryo.

[0077] Like at E16.5 expression of Medane is now restricted to theventricular and subventricular zone of the striatum (str), that is therostral migratory stream but can now also be found in the lower layersof the developing somatosensory cortex (str=striatum, cx=cortex,III=third ventricle). The significance of the expression of Medane inthe lower layers of the somatosensory cortex, which persists intoadulthood remains unclear.

[0078]FIG. 10

[0079] Expression of Medane in coronal sections of and adult mouse.Expression of Medane can now only be found in dispersed cells in thelower layers of the somatosensory cortex (not shown) and in single cells(arrows) around the third ventricle (3^(rd)) and the olfactory ventricle(ov), the reminiscent of the rostral migratory stream of embryonicdevelopment and the location of neuronal stem cells. Specifically thislocation supports the idea, that cells expressing Medane are theprogenitors of the TH-expressing interneurons of the olfactory bulb,which are generated persistently during adulthood.

[0080]FIG. 11

[0081] Ectopic specification of TH-expressing cells by Mdn in Zebrafishembryos. a) control embryos injected only with GFP RNA showingTH-expressing cells in the lateral midline of the diencephalon. b+d)Lateral views (b=right and d=left) of injected embryos with Mdn RNAshowing a 10 fold presence of ectopic TH-expressing cells in the lateralmidline of Diencephalon. c) A new cluster of cells with neuronalmorphology expressing TH are shown in the ventral midline of thediencephalon in an injected embryos with Mdn RNA.

[0082]FIG. 12

[0083] Expression of Medane and TH in the neuroepithelium. Expression ofMdn and TH in the neuroepithelium at E12. Note that Mdn expression canbe found in cells close to the ventricular surface, in the ventricularzone (VZ), whereas TH expressing cells are found more distal to theventricular zone in the differentiating zone (DZ) of theneuroepithelium. The expression domains overlap in the differentiatingzone. This spatial distribution of the two expression domains fits thehypothesis that Mdn is expressed in dopaminergic neuronal precursorcells which then migrate out of the ventricular zone whiledifferentiating thereby taking on the dopaminergic-TH-expressingphenotype.

DETAILED DESCRIPTION OF THE INVENTION

[0084] Genomic Structure of Mdn

[0085] As a result of the searching for new transcription factorsspecifying dopaminergic neurons, we have performed RT-PCR withdegenerate primers from the conserved bHLH domain of hairy/E (spl)related genes. Sequencing results shows the PCR-generated insert of H2clone was derived from the mRNA of a new bHLH protein. Database searcheswith the deduced amino acid sequence revealed that the predicted bHLHregion of clone H2 is a novel protein and related to proteins of theDrosophila hairy and E (spl) family.

[0086] The Mdn transcript is distributed over 4 exons and the gene spans1000 kb of genomic DNA. The genomic organisation of Mdn is shown inFIG. 1. All the introns were located within the coding region. Analysisof the DNA sequences at the intron-exon boundaries of Mdn showed thatthey all adhere to the 5′gt/ag3′ splice junction consensus rule fordonor and acceptor splice sites (Breathnach and Chambon, 1981; Shapiroand Senapathy, 1987). Southern blot analysis of total mouse genomic DNAdigested with HindIII showed a single band when hybridised with thefull-length cDNA of Mdn, under non-stringent conditions. Therefore, Mdnis a single-copy gene per haploid.

[0087] The exons of MDN range in size from 97 to 687 bp and the size ofthe introns vary from 220 to 453 bp. Primes were designed from intronicsequences to allow amplification of each exon. The amplificationproducts were designed to be fewer that 400 bp in length in order tofacilitate their use in SSCA/heteroduplex protocols for mutationalanalyses. The primer sequences, product sizes, and annealing conditionsare shown in Table 1.

[0088] A single start site was detected for Mdn when RACE and cDNAlibrary screening were performed as described in the Examples. Wedesignated this nucleotide the start (+1) of the transcript. We werenever able to obtain a product when primers MdnPr1D or MdnPr2D (20-100bp upstream of the transcription start site, respectively) were used incombination with primer H2.10R (exon 2) on RNA template. Therefore, itseems unlikely that there is any RNA species containing these sequences5′ of our designated first exon.

[0089] Computer analysis of the region usptream of the transcriptioninitiation site of Mdn transcript predicts a TATA box (position −36).1500 bp sequence between the 5′ proximal region of transcription startsite and part of intron 1 of Mdn there is a GC-rich region containingthree stretches of DNA that satisfy the criteria for CpG islands werefound (a ratio of observed/expected CpG>0.6 and a content of G+C>50. Asearch of the 5′-flanking regions of Mdn for cis-acting regulatoryelements revealed three putative Spl-binding sites. There were also fourrecognitions sites for N-boxes, six sites for E-boxes (class A, B and C)and one for RBP-JK (direct target of Notch). Concerning the 3′ UTR ofMdn, two canonical polyadenylation signal sequences AATAAA were present21 and 46 bp upstream of the polyadenylation site.

[0090] Primary and Secondary Amino Acid Structure of Mdn/MDN

[0091] Mdn/MDN cDNAs contain a 723 bp open reading frame starting fromthe first ATG codon present at nucleotide residue 85. Since it is theonly ATG codon upstream the bHLH, the first Methionine is assigned asthe initiation codon.

[0092] Mdn encoded a proteins of 241 amino acids residues in length, andthe calculated molecular mass is estimated to be 27.042 kD. The bHLH ofMdn (amino acid residues 11-59) shows 57/63% similarity and 49/43%identity to the hairy/E (spl) gene products, respectively (FIG. 2).

[0093] Two additional helices (III and IV, referred to as “orangedomain”) present in hairy and E (spl), were also found in thecorresponding position of Mdn. Comparison analysis between Mdn and MDNproteins shows a similarity of 93.4% and an identity of 91.4%. Thebipartite nuclear translocation signals, the bHLH domain, the orangedomain, and the proline rich region as well (20% residues encoded by theportion between amino acid residues 132-242) are conserved between humanand mouse Medane genes. During sequencing, we identified an amino acidpolymorphism in the coding region of Mdn: aa Pro156 (CCG or CCA).Another polymorphisms were detected in the 3′UTR: nt 838 (C or T). Thesechanges may be useful as allelespecific polymorphisms for use in linkagedesequilibrium studies.

[0094] Since Mdn protein harbours a putative bipartite nucleartranslocation signal in its N-terminal region, we further attempted toevaluate the functional significance of this putative domain. A humancell line were transient transfected with a GFP-tagged Mdn-codingvector. Control transfections with the blank vector (GFP) resulted. Thesignal was observed throughout the cell while cells transfected withMdn-GFP recombinant protein showed a fluorescence signal only in thenucleus (FIG. 3).

[0095] Mapping Data

[0096] Results of mapping with the T31 Radiation Hybrid (RH) panellocalized Mdn 15.9 cR from marker D8Mit297 on mouse chromosome 8,between markers D8Mit297 and D8Mit98.

[0097] Concerning the human gene, framework mapping of MDN was firstestablished by G3 mapping panel. This studies places MDN on humanchromosome 4 between markers WI-4886 and AFMA239XA5. Given theimportance of genes mapping to telomeric positions, to achieve an evenmore precise localization, we performed a G3 RH mapping panel. This lastpanel mapped also MDN in the telomeric region of human chromosome 4,between markers (SHGC4-3 and SHGC-63497). Moreover, both mapping datafor Mdn and MDN loci in mouse and human chromosomes, respectively,agrees with the syntenic region established for these two chromosomalpositions.

[0098] Expression Pattern of Mdn

[0099] In order to analyse the expression pattern of Mdn in thedeveloping embryo and in the adult we performed in situ hybridisation onwhole embryos (E9.0-E11.5) and on sections (E12.5-adult). Expression ofMdn is nearly exclusively restricted to the CNS. The only otherexpression domains are the vomeronasal organ, dispersed cells in theolfactory epithelium and in adult testis (FIG. 4).

[0100] To determine whether Mdn is indeed expressed in the dopaminergicneuronal precursors, its expression was analysed in various mousetissues, then studied the correlation with the expression of TH (ahallmark protein of catecholaminergic system). Transcription of Mdn isfirst detected at low levels at E9. At E9-10.5 mouse stages thetranscript is restricted to the developing ventral mesencephalonanterior to the isthmic organiser (FIG. 5). This is the region where 2.5days later dopaminergic neurons will develop, as determined by thepresence of TH+cells.

[0101] During the subsequent development of the CNS further Mdnexpression domains can be found. At E12 (FIG. 6) Mdn is still highlyexpressed in the ventral mesencephalon, the future substantia nigra(sn). However, now it can also be found in the dorsal part of the 3^(rd)ventricle, the hypothalamus, the developing striatum and in dorsal rootganglia. All these regions are characterized by the onset of tyrosinehydroxylase expression around this time (FIG. 6). Interestinglyexpression of Mdn starts to cease in these domains once TH-expressionbecomes prominent. E.g. at E14.5 (FIG. 7) TH-expression is strong in thesubstantia nigra (sn) but now Mdn expression is absent in this region.During further development Mdn also ceased to be expressed in all otherexpression domains except in the subventricular zone (SVZ) (FIGS. 8, 9),a germinal region which continuously generates new neurons destined tothe olfactory bulb even during adulthood (Temple and Alvarez-Buylla,1999). Neurons from the subventricular zone migrate along the rostralmigratory stream (RMS), then differentiate into local interneurons andfinally reach the olfactory bulb (Luskin, 1993, Lois and alvarez-Buylla,1994; Doetsch and Alvarez-buylla, 1996), where a large part of themdifferentiate into TH-expressing neurons. At E16 and E18 Mdn expressionis very prominent in the RMS, however, not in the olfactory bulb. Inadult brain, Mdn can still be found in dispersed cells in the lowerlayers of the somatosensory cortex and in single cells around the 3^(rd)ventricle and the olfactory ventricle (FIG. 10) representing thelocation of neuronal stem cells and the RMS. Thus, during developmentand in adulthood Mdn is expressed only in regions where subsequently THpositive dopaminergic neurons will arise.

[0102] Ectopic Expression of Mdn in Zebrafish

[0103] The correlation between Mdn and TH expression suggested that Mdncould specify DA neurons. To test this hypothesis, we expressed Mdnectopically in the zebrafish. After injection of capped Mdn RNA into16-cell zebrafish embryos, we found a 2-10 fold increase in the numberof TH-expressing cells in the normally TH positive diencephalon cluster(100% of cases, n=20), but also new cluster of cells with neuronalmorphology that express TH were found in the ventral midline of thediencephalon (FIG. 10). To discard the possibility that injection of Mdninduce general neurogenesis by mimicking the function of other bHLHrather than specific induction of DA neurons, we tested for ngnlexpression by in situ hybridisation. No general induction ofneurogenesis was detected in any embryo.

[0104] The following examples are set forth for illustrative purposesand should not be considered as limiting the scope of protection of thepresent invention.

EXAMPLES

[0105] Cloning of Mdn Gene Transcript.

[0106] A clone containing a partial Mdn cDNA resulted from theapplication of RT-PCR approach using degenerated primers.

[0107] The mouse foetal cDNA source was prepared as follows: ventralpart of the midbrain from mouse embryonic day 8-13.5 (E8-13.5) was mixedprior to RNA isolation. In parallel experiments, RNA from the rest ofthe brain and body was isolated as well. Total RNA was extracted usingRNeasy Mini Kit from Qiagen (according the manufacturer'srecommendations) followed by poly(A)⁺ RNA selection, using DynabeadsOligo (dT)₂₅ (Dynabeads mRNA purification kit).

[0108] Furthermore, first-strand synthesis is carried out by MoloneyMurine Leukemia Virus (M-MuLV) reverse transcriptase (AmershamPharmacia). After an annealing step of 5 minutes at room temperature ofthe poly(A)⁺ RNA from the mentioned tissues with an oligo pd (N)₆, thereaction is performed at 37° C. for 1 hr. This single strand cDNA wasused as a template to carry out an RT-PCR with degenerated primers. Theprimers were designed on the basis of the conserved amino acids from thebHLH domain of Drosophila hairy and its related protein her5 ofzebrafish (Müller et al., 1996). Consequently, fully degenerate primersdirected to conserved amino acids from the basic-helix-I of hairy(RRRARIN) and from her5 (RRRDRIN) proteins, as well as reverse primersfrom helix-II of hairy (EKADILE) and from her5 (EKAEILE) were designed.The third codon positions of fourfold degeneracy were substituted byinosine. Combinations of forward against reverse degenerated primerswere subjected to PCR in different experiments. Briefly, cDNA from theventral part of the midbrain and from the rest of the brain and body aswell of a mouse embryo E9-10 were used as a template in parallelexperiments. Conditions for the hot-start PCR were 94° C. for 4 min,then 95° C. for 30 s, 52° C. for 1 min, 72° C. for 50 s for 32 cyclesfollowed by a 5-min extension at 74° C. in 50 μl. PCR products werepurified through RCR-column (Qiagen) and then one-fiftieth of the firstPCR products were subjected to a second round of amplification of 33cycles at 60° C. of annealing temperature. In this second PCR, an EcoRIand a XbaI site was added to the 5′ forward and reverse degenerateprimers, respectively, for further subcloning in pBluescript vector. PCRproducts were electrophoresed on a 1.5% agarose gel. The cDNAs amplifiedfrom the ventral part of the midbrain were compared with the cDNAsobtained from the rest of the E9-10 embryo. Those that appeared to bespecific and unique for the ventral part of the midbrain weresubsequently double cut with EcoRI and XbaI restriction enzymes, thenpurified by phenol-chloroform extraction and finally subcloned intoEcoRI-XbaI of pBluescript vector. Colonies were gridded in duplicateonto nylon filters. To detect clones containing a bHLH related to hairyor her5 proteins, the filter was hybridizised with an oligomer coveringthe bHLH domain of hairy or her5, respectively. A gradient of stringencywashes in distinct experiments was done to detect those clones withhigher similarity to the hairy or her5 bHLH domain. Positives cloneswere sequenced with M13D and M13R primers using fluorescent DyeDeoxyTerminators on an ABI373A automatic DNA sequencer (Applied Biosystem).

[0109] As a consequence of the RT-PCR approach, we detected a positiverecombinant clone (clone H2). This new clone contained a novel EST,which turned out to be a partial cDNA of a new gene encoding a bHLH wecall Medane (Mdn). The sequence data of the mouse H2 clone are asfollows: 5′agaaggagagaccgaattaaccgctgcttgaacgagctgggcaagacagtccctatggccctggcgaaacagagttccgggaaactggagaaggcggagatcctggag3′

[0110] Full-length cDNA of Mdn Gene

[0111] The partial cDNA obtained from clone H2 was then used to obtainthe full-length of Mdn transcript in parallel approaches: a) Rapidamplifications of cDNA ends (RACE). 5′ RACE was carried out using theMarathon-Ready cDNA Amplification Kit (Clontech) from mouse 11-dayembryo, with the Mdn-specific primers H2-9R and nested primer H2-8R. Theproducts were subcloned into pBluescript TVector for sequencing.T-vector was prepared essentially as described by Marchuk et al.,(1991). 3′ RACE was performed according to Frohman, (1993) using primerQT₂₀ in the first-strand cDNA synthesis reaction from mouse 9-10-dayembryo poly(A)⁺ RNA. The cDNA products were submitted to cycleamplification. PCR was carried out using Q₀ primer and the Mdn-specificprimer H2.1. A second round of PCR was performed with Q₁ and nestedspecific primer H2.3. Products were subcloned in pBluescript TVector andsequenced. Clone 200A reveals the 5′ UTR whereas clone H2-A4 reveals the3′ UTR of Mdn; b) cDNA library screening: Approximately 4×10⁶recombinant phages from a mouse 11-day embryo cDNA library in lambdaTriplEx vector (Clontech), were screened with 873 bp PCR product derivedfrom clone H2-A4 containing the 3′ part of the coding region and the3′UTR of Mdn as well (base pairs 166-1000). Hybridisation was in 0.5 Msodium phosphate, pH 7.2/7% SDS, at 65° C. overnight. Membranes werewashed with 2× SSC/0.5% SDS for 15 min at 45° C., 1× SSC/0.5 SDS for 30min at 65° C., and 0.2× SSC/0.5% SDS for 30 min at 65° C. Three isolatedphages revealed to contain the putative full length of Mdn transcript.

[0112] To identify the human homologue of Mdn gene, approximately 8×10⁶recombinant phages from a human foetal brain CDNA library in lambda gt10(Clontech) were screened with the 873 bp PCR product described above.

[0113] The conditions of hybridisation and washing were also as describeabove. Two positive clones containing the cDNA of MDN were found bysequencing.

[0114] Database Search

[0115] The recent publication of the entire heterochromatic sequence ofDrosophila melanogaster and C. elegans, allowed us to look for thecounterpart of Mdn gene in this organisms. We used the BLAST search(Altschul et al., 1997) programs available onhttp://www.ncbi.nlm.nih.gov to look for the ortologue of Mdn inDrosophila and in C.elegans databases. The nucleotide and amino acidsequences corresponding to the bHLH domain of Mdn were used as a query.

[0116] Exon Identification and Amplification

[0117] BAC genomic clones containing Mdn/MDN loci were isolated byscreening of a Mouse/Human BAC genomic library (Resource Center of theGerman Human Genome Project-DHGP) when a partial cDNA of Mdn/MDN (basepairs 166-1000) was used as a probe, respectively. BAC-DNA preparationfrom the positive BACs were obtained and prepared for direct sequencing.Intron-Exon boundaries were identified and the precise lengths of theintrons of the mouse and human Medane gene were determined by comparingthe Mdn/MDN cDNA sequences to the genomic DNA sequences obtained from amouse/human Medane-containing BAC, respectively. To check theintron/exon structure of Mdn/MDN, we designed a set of primers coveringthe entire cDNA. Combinations of primers that have identical sized bandsusing the cDNA and genomic DNA as templates indicated a lack of anintervening intron between the two primers. The presence of an intronwas indicated by a discrepancy in the size of PCR product produced byusing genomic and cDNA templates. Those PCR products with primercombinations that indicated the presence of an intron were then used forsequencing in order to identify the splice donor and acceptor sites. Inorder to allow mutational screening of MDN gene, intronic primers weredesigned to amplify each exon (table 1), tested on human genomic DNA andthe products sequenced with nested primers. PCR was performed in 50 μlvolumes with 200 ng of human genomic DNA, 10 pmols of each primer, 1.25μM dNTPs, 1.5 mM MgCl₂, 1 U of Taq polymerase (Fermentas) and 6% DMSO.PCR amplifications were performed in an eppendorf thermal cycler, usinga hot-start procedure. Initial denaturation of samples was at 95° C. for4 min followed by 33 cycles at 56° C. annealing temperature for all PCRprimer pairs.

[0118] Whole Mount in Situ Hybridisation

[0119] Pregnant mice were killed by cervical dislocation, embryos weredissected, fixed overnight at 4° C. in 4% paraformaldehyde. Fixedembryos and brains from different stages (E8.5-E18) were treated andwhole mount in situ hybridisation were performed as described (Sporle etal., 1998). Antisense and sense digoxigening (DIG)-labelled riboprobesfor Mdn (base pairs 166-1000) were produced using a DIG-RNA labellingkit (Boehringer-Mannheim), following the manufacturer's instructions.

[0120] Histological Analysis

[0121] Brains of embryonic mice or whole embryos were eithertranscardially perfused or immersion fixed overnight at 4° C. in 4%paraformaldehyde. Some of the adult brains were shock frozen on dry ice.Perfused brains were either cut on a cryostat in 30 μm thick sections orparaffin embedded and cut on a microtome in 4-8 μm thick sections.Frozen brains were cut on a cryostat in 18 μm thick sections andprocessed for in situ hybridisation. in situ hybridisation of frozen andparaffin sections was performed after a modified method of Dagerlind etal. (1993). Antisense and sense mRNA probes transcribed from linearizedplasmids containing fragments of TH (base pairs 23-788), and Mdn (basepairs 166-1000) were used as a probe. Following in situ hybridisation,sections were counterstained with cresylviolet according to a modifiedmethod by Nissl (1894).

[0122] Northern Analysis

[0123] Poly(A)⁺ RNA from mouse 8.75-15-days embryos was prepared asdescribed above. 2.5 μg of poly(A)⁺ RNA and 3 μg of RNA ladder (0.24-9.5kb RNA Ladder; GibcoBRL) were electrophoresed on a 1.2%agarose/formaldehyde gel and then transferred onto a nylon membrane(Hybond-N⁺, Amersham Pharmacia) in 20× SSC. The filters were hybridisedovernight at 65° C. in 0.5 M sodium phosphate buffer, pH 7, 2/7% SDSwith a partial Mdn cDNA probe (base pairs 166-1000). To check equalloading of RNAs, Northern were reprobed with the GAPDH CDNA. Themembrane was washed with 2×SSC/0.5% SDS for 15 min at 45°C, 1× SSC/0.5SDS for 30 min at 65° C., and 0.5× SSC/0.5% SDS for 20 min at 65° C. andexposed to X-ray film with intensifiers for 7 days. The mouse (Origene)and human (Clontech) adult multipletissue Northern blots containing 2 μgof poly(A)⁺ RNA from the tissues indicated were hybridised with a probecontaining a partial cDNA fragment of Mdn/MDN cDNA, respectively (basepairs 166-1000). Hybridisation was performed at 65° C. for 1.5 hr inExpressHyb hybridisation solution (Clontech), according to the protocolprovided. The membranes were washed twice in 3× SSC/0.1% SDS at 37° C.for 20 min, and then in 2× SSC/0.1% SDS at 50° C. for 20 min and 1×SSC/0.1% SDS at 65° C. for 10 min, and exposed to an X-ray film withintensifiers for 5 days. To check equal loading of RNAs, all Northernblots were reprobed with a GAPDH cDNA, with an exception for the mouseadult Northern blot which was reprobed with a β-actin cDNA.

[0124] Southern Blot

[0125] Mouse genomic DNA was digested with Hind III and electrophoresedon 0.8% agarose gel, then blotted onto nylon membrane (Hybond-N⁺,Amersham Pharmacia). Finally hybridizised with the entire cDNA of Mdn.Conditions for hybridisation and washing were identical to describedabove.

[0126] Radiation Hybrid Mapping Panel.

[0127] The radiation hybrid (RH) 100 cell lines DNAs of the T31mouse/hamster panel (Jackson laboratory) were tested plus parentalcontrols. A 212 bp fragment was amplified by PCR using forward primerH2.18D and reverse primer H2.19R from the first intron of Mdn to enhancespecificity. PCR was performed under standard conditions in 50 μlvolumes with five μl of RH DNAs. The PCR cycling profile was: 94° C. 3min, (94° C. 30 s, 55° C. 35 s, 72° C. 30 s) 40 times, 72° C. 7 min, 4°C. hold. In all cases, the hamster background gave rise no product,making scoring unambiguous.

[0128] Since radiation hybrid mapping is a +/− PCR assay and falsepositive and false negative reactions may distort the linkage, each cellline was typed twice. Any line that give a new (−) in a string ofpreviously linked (+), or vice versa, were retyped to determine thefinal correct score for each cell line. Hybrids that gave a signal inboth PCR reactions were scored as positive and those giving no signal inboth as negative. PCR products were electrophoresed using very sensitivedetection conditions on agarose gel and the data were analysed aspositive, negative, but not missing, since all the lines were checkedusing GAPDH primers. Results were submitted to the Whitehead mouse RHmap website for automated mapping data analysis.

[0129] Concerning the mapping of the human gene, the Stanford G3 mappingpanel of 83 RH clones of the whole human genome was used to map the MDNlocus. Primers used were H5D and H4R. Conditions and analysis werecarried out as described above. A server for the chromosome localizationof MDN was used at http://www-shgc.stanford.edu

[0130] The Genebridge 4 mapping panel of 93 RH clones of the whole humangenome was also performed, using the conditions described above.Chromosome localisation of Mdn was performed by accessing the serveravailable at http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl

[0131] Subcellular Localization of Mdn

[0132] A Mdn-GFP fusion protein was created by subcloning the codingregion of Mdn into pEGFP-N1 vector (Clontech) so that it is in framewith the EGFP coding sequences, with no intervening in-frame stopcodons, allowing the localisation of the fusion protein in vivo. Humanembryonic kidney 293 cell line (Graham et al., 1977; ATTC-Nr.CRL-1573)were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% foetalcalf serum and 1% Pen/Strep. 30-40% confluence plates of exponentialgrowing cells were transfected with 0.5 μg Mdn-GFP fusion protein usingstandard transfections methods with 2.5 M of calcium phosphate (Graham FL and Eb A J van der, 1973). Control transfections were carried out with0.5 μg of blank vector (pEGFP-N1). The following day, the subcellularlocalization of the recombinant fusion protein and control wasvisualized by direct fluorescence of intact cells.

[0133] Injection of Mdn in Zebrafish Embryos

[0134] For this, 16 cell-stage zebrafish embryos were injected with Mdncapped mRNA and the presence of ectopic TH-expressing cells wasdetermined 16 hr later. Capped mRNAs of Mdn were synthesised (Ambion),then verified by in vitro translation (Ambion) and the translationproduct was checked in a SDS-PAGE gel. Embryos were obtained fromnatural spawning of wild-type adults. Injections were carried out intoone central blastomere of the 16-celled embryo (50 pg), together withthe lineage tracer GFP MRNA (50 pg). 12 hrs after injection, thepresence of Mdn RNA was checked by in situ hybridisation of fixedembryos using a antisense cDNA of Mdn as a probe (base pairs 166-1000).Only embryos having received the injection in the CNS (sorted out at 16hrs under fluorescence) were analysed. Phenotypic analyses, in situhybridisation and immunocytochemistry were done following standardprotocols (Hauptmann and Gerster, 1994). For the immunodetection of TH,a polyclonal anti-TH antibody (Chemicon) was used at 1/1000. Fordetection of neurogenin1 (ngn1) RNA, a ngn1probe was used (Blader etal., 1997).

[0135] Knock-out Targeting Strategy (Animal Model)

[0136] Firstly, a targeting vector containing a mutated allele of Medaneis designed to recombine specifically with the Medane locus. Thecomponents of such a vector are sequences which are homologous with thedesired chromosomal integration site of Medane. For the generation ofthe 5′ arm, a fragment of isogenic homologous sequences of 1758 bp wasused, including also the first exon and the first intron. The 3′ arm wasa SmaI-Eco47III fragment (5245 bp) containing the 3^(rd) exon and theentire 3′ UTR of Mdn. Other components were also included in the vectorsuch as the beta-galactosidase gene (lacZ) as a reporter gene for theMedane expression, PGK neo gene (neomycin phosphotransferase) as apositive marker, and the PGK tk gene (thymidine kinase) as a negativeselector markers. These markers provide strong selection for the clonesthat have been targeted in the right locus by homologous recombinationevent. The neo marker was surrounded with lopxp sites to allowsite-specific recombination in mice. This technique makes possible togenerate a germline mutation without the positive selection marker usingCre-loxP system. The vector is linearized outside the homologoussequences before transfection into ES cells. To confirm that the desiredgenetic change has occurred, a diagnostic Southern screening strategywas designed. Therefore, to analyse both the 5′ and 3′ aspects of thetarget locus, we used 5′ and 3′ external probes from sequences flankingboth ends of the homologous sequences. Pluripotent embryonic stem (ES)cells derived from a mixed 129SV background were electroporated with thetargeting vector for introducing the mutated Medane allele into EScells, then plate them under double selection (Gancyclovir and G418) infeeder plates. Homologous recombination events were detected bygenotyping using BamHI digestion and southern blot analysis. The finalrecombinant allele (Medane is mutated) raised from the desired geneticexchange as a consequence of double reciprocal recombination event whichtakes place between the vector and the chromosomal sequences. In thisway, the wild type Medane allele is replaced by all the components ofthe vector which are between the 5′ and 3′ homologous sequences. Theheterologous sequences at the ends of this arms of homology are excisedfollowing targeting. In this way, a mutant cell is created lacking thesequence of the gene encoding for the nuclear translocation signal, thebHLH domain of Medane, and the second intron as well.

[0137] Blastocyst recovered from pregnant superovulated females wereinjected with the Medane-mutant ES cells and transferred into apseudopregnant host female. Germ-line transmission is now determined byPCR and Southern blot analysis of tail DNA. Chimeras were bred withC57BL mice to obtain F₁ offspring. Heterozygotes (Mdn^(+/−)) for thetargeted allele can be mated together to produce F₂ litters withwild-type (Mdn^(+/+)), heterozygote (Mdn^(+/−)), and homozygotes(Mdn^(−/−)) for analysis.

[0138] Primers and Probes H2.1: (5′-tcgctgcttgaacgagctg-3′); H2.10R:5′-cagagttccgggaaactg-3′; H2 18D: (5′-gagactggaaggagagtcc-3′); H2.19R:(5′-agggtcactaattcgccaac-3′); H2.3: (5′-tggcaagacagtccctatgg-3′); H4R:(5′-ctggttccacctccttctc-3′); H5D: (5′-ccgctagaagttctgctgg-3′); MdnPr1D:(5′-ggagccccctcggacct-3′); MdnPr2D: (5′-caaacgcagaactcctaatcc-3′);MdnPr1D: (5′-ggagccccctcggacct-3′); MdnPr2D:(5′-caaacgcagaactcctaatcc-3′)

[0139] It is still a major challenge to understand how dopaminergicneurons are specified and assigned their fate in the vertebrate CNS. Ina search for bHLH-containing transcription factors that might functionas intracellular mediators for the specification of dopaminergicneuronal lineage in the vertebrate CNS, we have isolated, characterisedand mapped a new murine gene and its human counterpart. The cDNA of thenew gene, we termed Medane (Mdn) (for Mesencephalic Dopaminergic neuronsE (spl) and hairy related gene), encodes a bHLH protein related to theproducts of hairy and E (spl) genes of Drosophila.

[0140] Most of the genes governing the choice of neural fate frommultipotent progenitors cells in a variety of tissues and organisms(reviewed by Garrell and Campuzano, 1991) are bHLH proteins. There isevidence that mammalian homologues of Drosophila bHLH play and importantrole as a regulators of cell fate decisions in the developing nervoussystem and inducers of neuronal differentiation at the level of genetranscription (Reviewed by Jan and Jan, 1993; Lee, 1997).

[0141] Mdn protein share high structural similarities in the bHLH regionwith several cDNAs encoding proteins of the hairy-E (slp) family (FIG.2) that have been cloned in mice, rat and human, including HES (Akazakaet al., 1992; Sasai et al., 1992), SHARP (Rossner et al., 1997), HRT(Nakagawa et al., 1999), DEC1 (Shen et al., 1997) subclasses, but havecharacteristics that are distinct from those mentioned above. Mdndiffers from Hairy/E (spl) and HES transcription factors by the absenceof both the proline residue in its basic DNAbinding domain, and thecarboxy-terminal WRPW amino acid motif. In both Drosophila andvertebrates, these features have been proposed to confer unconventionalDNAbinding specificity to bHLH proteins and to permit the recruitment ofGroucho-like cotranscriptional repressors, respectively (Fischer andCaudy, 1998, and references therein). We specifically note Mdn isdivergent in several critical and conserved amino acid positions in thebHLH domain characteristic within the SHARP, HRT, HEY and DEC subclass.In addition, the full-length transcript of Mdn shows that the similaritydoes not extents into the N-terminus and C-terminus of other hairy/E(spl)-related genes. This observation argues in favour that Mdnconstitute a new subclass of bHLH transcription factor distinct andclosely related gene of hairy and E (spl). Then, we termed the gene asMedane to emphasise the distant features of this new gene and thepreviously cloned mammalian hairy-E (slp) related proteins.

[0142] Expression of Mdn mRNA is detected in a very dynamic pattern inthe embryonic CNS. To analyse the tissue distribution and theontogenetic expression pattern, we performed whole mount in situ andcryosections experiments. Transcription is first detected at low leveland during early embryonic mouse 9-day (E9) strikingly restricted to theproliferative neuroepithelium of the developing ventral mesencephalon,where dopaminergic neurons develop. This early starting appearance ofMdn in the ventral part of the midbrain coincides with the observationthat the progenitors for mesDA neurons also lie on this part of themouse embryo as early as E9 (Hynes and Rosenthal, 1999). The otherintracellular molecules that are known to be implicated in the mesDApathway, Ptx3 and Nurrl, start to be expressed in the mouse mesDAterritory at E10.5/E11, respectively. In contrast, Mdn is uniquelyexpressed in the mesDA system as early as E9, when the progenitors forthis neuronal cell type start to differentiate into DA (Hynes andRosenthal, 1999) and became TH⁺ cells at E11.5.

[0143] Interestingly, in contrast to Nurr1 and Pxt3 genes, which none ofthem are capable to induce dopaminergic fate in embryonic explants(Hynes and Rosenthal, 1999), Mdn is capable to specify DA neurons whenexpressed ectopically, may be activating a program for DA-specific geneexpression and differentiation. These data suggest that Ptx3 and Nurr1for a regulatory cascade for development of the mesDA system in whichMdn may act as an upstream activator.

[0144] As determined by in situ hybridisation with TH cRNA probe, MdnmRNA expression was detected in a spatially correlated distribution,although TH appears slightly later when differentiating cells arepresumed to have migrated further away. Detailed analysis of in situhybridisation experiments on consecutive sections either incubated withthe ³⁵S-Medane probe or the ³⁵S-TH-probe also revealed a spatialcorrelation between Mdn and TH expressing cells. Whereas Mdn isexpressed predominantly close to the ventricular surface where neuronalprogenitors are located, TH expression can predominantly be found incell layers more distal to the ventricular surface, the differentiatingzone (DZ). A layer of overlap between the two expression domains can befound (FIG. 12). This supports the idea that Mdn is expressed indopaminergic precursor cells but once these cells start to differentiatefurther they migrate away from the ventricular surface and lose Mdnexpression. The expression of Mdn in the RMS but not in the olfactorybulb during late embryonic development and around the 3^(rd) ventriclein adulthood also fits this idea: since once the cells have entered thebulb they enter a more differentiated state, e.g. start to express TH.

[0145] Taken together, the temporal-spatial expression pattern of Mdnstrongly suggests that it is expressed in dopaminergic precursor cellsduring development as well as in adult mice. Moreover, given the closeassociation between Mdn and TH expression in developing DA system andthe observation that Mdn expression does not overlap TH expression inthe adult mouse CNS, it is likely that Mdn is involved in thespecification but not in the maintenance of this subset of dopaminergicneurons. Taken together our results about the expression of Mdn, it islikely that Mdn is rather involved in the early events of development ofthe mammalian DA system, but Nurrl and Pxt3 in the late phases. Earlysteps include the generation of the appropriate numbers of neuronal andglial precursors and the migration of precommitted cells to their finalposition while late events encompass axonal outgrowth, dendriticarborisation, synaptogenesis.

[0146] Given the fact that expression of Mdn in zefrafish triggered theaparition of ectopic TH-expressing cells, not only where this neuronsare, but also in locations of the zefrafish CNS where TH-expressingcells are absent, suggests Mdn can function in a cell-autonomouslymanner. These results agrees with previous observations that Hairy/E(spl) factors and related genes, generally act cell-autonomously onprecursors in the regulation of cell specification (Fischer and Caudy,1998; Bally-Cuif et al., 2000). Although the action of manytranscription factors is likely involved in a given neuronalspecification, the present invention indicates that Mdn can function asa single activator of transcription required for the initial cell fatespecification of the mesDA cell identity and single bHLH may determinesingle neuronal cell identity.

[0147] Members of the hairy-E (spl) family of bHLH proteins have beenshown to be upregulated in vertebrate cells by Notch signalling. Aninteresting feature found in the third intron of Mdn is the repeat(GTT)₈(ATT)₉, which is characteristic of those genes controlled byNotch. Analysis of the sequences upstream the transcription start siteof Mdn, reveals also canonical target sites for Notch (RBP-JK),proneural genes (Box E-Class A), and for E (spl) (Box E-Class B) Takentogether, these observations suggest Mdn may also belong to the Notchsignalling pathway, suggesting that Mdn can links early patterningevents Notch-mediated to the differentiation of defined neuronalprecursors into dopaminergic fate. Despite the recent identification ofseveral bHLH hairy-related genes, the unique expression pattern of Mdnsuggests a previously unrecognised role for hairy-related genes in mesDApathway.

[0148] Despite of Notch-E (spl) network has been conserved in evolutionas a way to assign specific fates to members of groups of initiallyequivalent cells (Chitnis et al., 1995), we failed to found anyortologue of Mdn when Drosophila cDNA libraries were screened with a Mdnprobe. In addition, no matches were found when the nucleotide and aminoacid sequences of the bHLH from Mdn was blasted against Drosophila andC. elegans genome sequences, which the entire heterochromatic sequenceare already completed. These observations argue in favour of theemerging concept that bHLH proteins can insure the determination ofsubsets of neuronal phenotypes, and the apparition of new transcriptionfactor genes is causally linked to the appearance of new subclasses ofneurons during evolution. Then, Mdn, Hairy and E (spl) genes couldoriginated from the same or closely related ancestral genes, but Mdn hasoriginated later without homologue in Drosophila.

[0149] Since the cathelolaminergic system is large involved in humanneurodegenerative disorders, p.e. Parkinson's disease, theidentification of mouse genes controlling developmental mechanisms ofthese neurotransmitters, with a special regarding to the mesDA neurons,should provide new insights into the etiology of these disorders. Aspecific function of bHLH genes in the adult brain is not known, but anemergent concept is that neuronal bHLH proteins are also involved in the“adaptive” changes of mature CNS neurons, an a role in neuronalplasticity has been suggested (Bartholoma and Nave, 1994). Cells fromthe SVZ act as neural stem cells in both the normal and regeneratingbrain, and continually generate new neurons destined to the olfactorybulb. Since Mdn is expressed in the SVZ and follows the RMS until theolfactory bulb, we proposed Mdn can be involved also in thedifferentiation of adult stem cells of the SVZ into dopaminergicneurons, powering the regeneration of such population in the adultbrains. Thus, suggesting misexpression of Mdn due to its telomericposition on HC4 can cause a defective regeneration of dopaminergicneurons, and subsequently, a lack of DA neurons in adult brains.

[0150] Moreover, by expressing human proteins that specify the formationof specific types of neurons, it may be possible to generate neuronswith defined identities. Then, stem cells that have the capacity toself-renew and differentiate into neurons can be cultured anddifferentiated into DA neurons by expression of MDN.

[0151] Therefore, it is an aim of this invention to develop strategiesfor replacing neurons lost from disease or injury. Sinceneurodegenerative disorders such Parkinson's disease lead specificallyto the loss of DA neurons, an essential aspect of any neural replacementstrategy will be the ability to generate DA neurons. With this aim, thefunction of MDN may exploited, by expressing the gene in embryonic stemcells isolated from humans, to adopt them a DA neuronal fate.Manipulation of stem cells into dopaminergic cells in vitro could beperformed in preparation of tissue grafting and experimental therapy forParkinson's patients (Pogarell and Oertel, 1998; Sautter et al., 1998;Ahlskog, 1993; Defer et al., 1996).

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[0222] Further literature, to which reference is made in the descriptionby the respective numbers:

[0223] 1. Derossi D, Chassaing G, Prochiantz A: Trojan peptides: thepenetratin system for intracellular delivery. Trends Cell Biol 1998,8:84-87

[0224] 2. Lindgren M, Hüllbrink M, Prochiantz A, Langel U:Cell-penetrating peptides. Trends Pharmacol Sci 2000, 21:99-103

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[0230] 8. Schwarze S R, Hruska K A, Dowdy S F. Protein transduction:unrestricted delivery into all cells? Trends Cell Biol. 2000 July; 10(7): 290-5

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[0233] 11. Vives E, Brodin P, Lebleu B: A truncated HIV-1 Tat proteinbasic domain translocates through the plasma membrane and accumulates inthe cell nucleus. J Biol Chem 1997, 272:16010-16017

[0234] 12. Schwarze S R, Ho A, Vocero-Akbani A, Dowdy S F. In vivoprotein transduction: delivery of a biologically active protein into themouse. Science. 1999 Sep. 3;285 (5433): 1569-72 TABLE 1 PCR PrimersPairs for MDN mutation analysis FORWARD PRIMER NAME REVERSE PRIMER NAMESize Tm Exon sequence (5′ to 3′) sequence (5′ to 3′) (bp) (° C.)amplified MDN1D ctgatcttgaatgcatacatcc MDN1R cgggtcggtgagtcagatgc 311 561 MDN2D cccttcctagagcgaatctgag MDN2R gggcgtctccgcagagtgg 301 56 2 MDN3Dgcagggcgaacctcaggag MDN3R ctcgggaacactcagtcactcc 318 56 3 MDN4aDgtgccctgcacccctttgg MDN4aR aggcggccaggggaaagg 392 56  4^(a) MDN4bDggcgaggccgctgtgttcc MDN4bR ggagatccttcagaagactc 392 56  4^(b)

[0235]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 47 <210> SEQ ID NO 1<211> LENGTH: 1000 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (85)..(87) <223>OTHER INFORMATION: Start codon <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (808)..(810) <223> OTHER INFORMATION: Stopcodon <400> SEQUENCE: 1 gaacacttga agaggcacac gaggtggaac gtggacgagtgcctcgcgcc accgcctctg 60 ccgagggcgc gcacgcacac cagaatgtct gacaggctcaaggaacgcaa aagaaccccg 120 gtttctcata aagtgataga aaagagaagg agagaccgaattaaccgctg cttgaacgag 180 ctgggcaaga cagtccctat ggccctggcg aaacagagttccgggaaact ggagaaggcg 240 gagatcctgg agatgacagt gcagtacctc agagctctgcattccgcgga ttttccccgg 300 ggaagggaaa aagcagagct tttagcagaa tttgccaactacttccacta cggttaccac 360 gagtgcatga agaacctggt gcactacctc accaccgtggagcggatgga gaccaaagac 420 accaagtatg cgcgcatcct cgccttcttg caatccaaggcccgcctggg cgccgagccc 480 acctttccgc cgctctcgct tccggagcca gatttctcctatcagctgca tgcagcaagc 540 ccagagttcc cgggccacag cccaggtgaa gccacaatgttcccgcaggg ggctacccca 600 gggtcattcc catggcctcc tggagctgcc cgcagcccagcactgcctta cttgtccagc 660 gcgacggtac ctctccccag cccggcacag cagcacagccccttcttggc tccgatgcag 720 ggcctggacc ggcattatct caatctgatc ggccatggccaccccaacgg cctcaacctg 780 cacacgcccc agcaccctcc ggtgctctga cacccactcactgcctagat tactttgcga 840 ttcggtggcg gcttgagacg atgtatatgt tgtacgagtgtaaatagtgt gctaacaaga 900 tctggatggg aaagcgtcag aagcgaattc gccttctgaaggtcctcccc aaccaataaa 960 tatttttgtg ctaaagatca ataaatattt tgtgctaaag1000 <210> SEQ ID NO 2 <211> LENGTH: 983 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <400> SEQUENCE: 2 ggacactcga ggaggcacac gaggcggaaaagtggacggg tgccccgcgc caccgcctct 60 cccgagggcg cgtactgacc aggatgtcagacaagctcaa ggaacgcaaa agaacccccg 120 tttctcataa agtgatagaa aagcggaggagggacaggat caaccgctgc ttgaacgagc 180 tgggcaagac agtgcccatg gccttggcgaagcagagttc cgggaagctg gagaaggcgg 240 agatcctcga gatgaccgtt cagtacctgagagcactgca ctccgctgat tttccccggg 300 gaagggaaaa agaacttcta gcggagtttgccaactactt ccactatggc taccacgagt 360 gcatgaagaa cctggtgcat tacctcaccacggtggagcg gatggagacc aaggacacga 420 agtacgcgcg catcctcgcc ttcttgcagtccaaggcccg cctgggcgcg gagcccgcct 480 ttccgccgct gggttcgctc ccggagccggatttctccta tcagctgcac cctgcggggc 540 ccgaattcgc tggtcacagc ccgggcgaggccgctgtgtt cccgcagggc tctggtgccg 600 ggcctttccc ctggccgcct ggcgcggcccgcagccccgc gctgccctac ctgcccagcg 660 cgccagtgcc gctcgctagc ccagcgcagcagcacagccc cttcctgaca ccggtgcagg 720 gcctggaccg gcattacctc aacctgatcggccacgcgca ccccaacgcc cttaacctgc 780 acacgcccca gcaccccccg gtgctctgacgcccactcgc ccgccagatt tctcctcgct 840 ttgggcgctt ttaggagaaa tgctgtatatattgtacaca taatgtgtaa atattgtacc 900 ccaaaaatct gggctggggg aggcaaagagcgaatgagtc ttctgaagga tctccccctg 960 gtaataaacg ttttctgata aag 983 <210>SEQ ID NO 3 <211> LENGTH: 10207 <212> TYPE: DNA <213> ORGANISM: Musmusculus <400> SEQUENCE: 3 cctccccccc ccccatattt cgctttctaa aaccataaaaaagagatcaa attccctaac 60 ttcacgggac ccttttcagt gacaaatatt gatgcccaaagtgtctgcgc tctcccgccc 120 ccaaacgtta agaaaaggcg cccgcgagag ggggacataaaaagttaaca atgctgtgaa 180 aatatgtttg caaaaaatag acaatcgttg gattaaacgtattcaagtat gaaataatgc 240 ctttttgtgt caaaacttgg gcgatgggcg ggtacaaaagttccctgtgg cagctacttg 300 ctccctttgt gagctgtgcg ctttggcgtc tccacttgggcgcattaccc agagccctct 360 aagcgcgatt gtttctccct ttctaatgac atttaccggatcaaaacatg ctgttaattc 420 gatcagaagg cttcaccctc cctgacaaag ccacaataatttctcctgaa gtttgttaaa 480 ttgaccaaaa ttaggcaaat gaataggggt ctgtaggcgccccctcatgc aggtgacggc 540 gcataatgct cgcctgggcc agctgcattc tcccttttcttctcggccca ctctcctctg 600 tgttgcccca atcctcccac ccccctcaca cacacacacacacacacaca cacacacaca 660 cacacacccg cctgtctgtc ctccacccca ctcttggcattattaaaatt tagcccaatg 720 aaactattca tacttttcaa tggacatttt cctataggataataggctac aaattgagcc 780 tcttcccccc gcgaagagag agccggaggg gggagaaaagaaagcagagg atttggtcag 840 agactggggc ggggggagag agagtcgcgt gccaaaggcccgcgggaggg gcgaagcgag 900 gcgggagcct cgtgtgccag ccgcagcccc acacctgccgggatgtccgg acaaataaag 960 cggtgtaaac aaaaaggggg gagacggacg tgtcaccaagtcgtgtgaga aaagcctggg 1020 aacaaacggg gcgcctccgt ctccagaagc tctcccttgaacccggcgga acagcctatt 1080 aaaggcttac ttaattactt taatgactct ggacaggctttaaaacgcac tcggcgctgg 1140 gaccgcgggc ttgctgggat ttgtaaacag gcgatcgtgtgagactcagc ggtaggacta 1200 aaggaagcga cgctgttttg tgaaggtcct cgccccccggtgcccgcagc cattgcgccc 1260 ctgcgctctg cgccggctaa gagtgcaggc gctcgctggtccgccggctc cagtttctcg 1320 cccccttctt cctgaggtgc ctgttgccat gcaaatgttattcctggacg aatcacgtgt 1380 cctttgaaga gccacgtgga ttaacaaagc tgatctgcccccacctcgtc cccctcggtt 1440 gctaaaaaat tttttgtcaa cttctttaaa actgaccttgaatgcataca tgctccaaac 1500 gcagaactcc taatcctatg gaataattca gcgcgtgagaatgcacttgc agtcctagat 1560 ggctgcttta taaagggagc cccctcggac ctgggaggctgcagtctacc tggaacactt 1620 gaagaggcac acgaggtgga acgtggacga gtgcctcgcgccaccgcctc tgccgagggc 1680 gcgcacgcac accagaatgt ctgacaggct caaggaacgcaaagtgagtt ccttgcgctc 1740 tggtggtgga agagtgtgac tcgccaacta acctgacatctggctggata gatgactaag 1800 atgtgccacc cagaacttta agtgtggaga aatccagagtggtttggaaa gggaacagag 1860 actggaagga gagtccttga ttggagagag ctcactctgggaggcgggtg gccacgtccc 1920 agagccttgg cacttagctt ggccttggca gggatgtgtagggaagttga acggcaagag 1980 agccagccag cacggacaca gagctctgag ttccgggacacagagctctc ttggtctagt 2040 gtggcggggc aggggtctct tcgggttggc gaattagtgaacccttgtaa cacagaggct 2100 ttgccttttg gatgactcct tgggcgaagc catcttaaatgcaaaacttt ctgtttacag 2160 agaaccccgg tttctcataa agtgatagaa aagagaaggagagaccgaat taaccgctgc 2220 ttgaacgagc tgggcaagac agtccctatg gccctggcgaaacaggtaac gtttgtggtg 2280 ctgggtactc ccccactgtc actctctgca gagtccccaaagtctctaca gacgctgctg 2340 gcctaagaga actcgcccca ctctgggcgg cagtagcctctctgacgcca gcatcttcct 2400 ggctgctccc ctacagaaga cagagagcat gtgcgtgggtctgctgtgga tgggccgcct 2460 ctaaagctcc tgtggggttt ttgcagagtt ccgggaaactggagaaggcg gagatcctgg 2520 agatgacagt gcagtacctc agagctctgc attccgcggattttccccgg ggaagggaaa 2580 aaggtgggca ttgattcagg caaaggaaac ctgactggggagagtatgcc tcgcgggaac 2640 ctagggtctg agagtggagg acagtccggg agcaggaagcccccgaagcg tccaggcgct 2700 tggttcctga acatgcacct gctggatgct atggaccagcacctaaagat gcaggtctat 2760 tactcctcac aggcgtgggg tggcggggtc aggctcttgagacaagatta ctgcttttcc 2820 aatgtctaga ctgcagagta gagaatgcca atctcaaatgagaatccagc ccaaacccta 2880 ttaagatacc tcggcaccgt tctaggaagg ggtgtgcctgttctcttttg tttccttctc 2940 cctaccattt atgttgttgt tgttgttgtt gttgttattattattattat tattattatt 3000 attactttgc taccccagca gagcttttag cagaatttgccaactacttc cactacggtt 3060 accacgagtg catgaagaac ctggtgcact acctcaccaccgtggagcgg atggagacca 3120 aagacaccaa gtatgcgcgc atcctcgcct tcttgcaatccaaggcccgc ctgggcgccg 3180 agcccacctt tccgccgctc tcgcttccgg agccagatttctcctatcag ctgcatgcag 3240 caagcccaga gttcccgggc cacagcccag gtgaagccacaatgttcccg cagggggcta 3300 ccccagggtc attcccatgg cctcctggag ctgcccgcagcccagcactg ccttacttgt 3360 ccagcgcgac ggtacctctc cccagcccgg cacagcagcacagccccttc ttggctccga 3420 tgcagggcct ggaccggcat tatctcaatc tgatcggccatggccacccc aacggcctca 3480 acctgcacac gccccagcac cctccggtgc tctgacacccactcactgcc tagattactt 3540 tgcgattcgg tggcggcttg agacgatgta tatgttgtacgagtgtaaat agtgtgctaa 3600 caagatctgg atgggaaagc gtcagaagcg aattcgccttctgaaggtcc tccccaacca 3660 ataaatattt ttgtgctaaa gatcaataaa tattttgtgctaaagaaatg aagttctttg 3720 ctatcttttg ctcacccaca aaccatccac tctaagtgtcttacaaggaa tcctaagggg 3780 gctaggagcc caggtgaggg cattcagtaa tttagccaaggagagaggac ctttgcgcag 3840 tccgaggcag acagaatgga agcttaagca gatattagaggtgtctctta gggggtagga 3900 gactaaactc atggggtgct caagaccttc ctccttttatcctggccctg tgcgacatgt 3960 gcagaagtct ggaacctgac cttaagttct tggaaaatgcaaatcacaaa acaaccccga 4020 gaatggcagg agccctcttt tactagctgg gacctggaggcccttcctac tctcattggt 4080 ctggcaaagc gcaaagctca ggcaagccta atgaatagctgaggtcacag cctcaatatg 4140 tctctatttt aaaataaata aataaaataa aggcgcggagcaagaaacat tctaagttgt 4200 cttcgaagca tagtttgttc ttttcctccc ggtgacaatcactggacatc agaggggtcc 4260 cggactttcc cttttacgca caaatcaacc cctctgtagggctctagacg accctggcag 4320 atgcgcgcat atctgattgg aagatatgtg accttctcccccagggattc cccgctgatt 4380 ctgacacttg atccaaaagc tgagtcctct gttggggttctcaggtccgc ggagaaaggc 4440 tcagcccggc tctaaaatgg aatgttgctt ttaatctccctccgtgcaga gtgtggtgag 4500 aaaggttgtg tctagagagg gcgtggctgg tgttttttcacgcatttaca gatttttcag 4560 gagaccgcga tttttcaagg gatcagggtt ttcccttccggaggccgtgg gcagaccaca 4620 ctcccgtttt ttgctaaaat ccaaacagca aaatggacttctatgctctg ttaatatcaa 4680 caggtaaaat aaagcatcta accaggtgaa atagtggaaactgatttttt tcatcattaa 4740 ttttccacct ttagaatgtt ggtcctgtta aaattggtgccctttaaaca agtaatttga 4800 tattatgtca tattgttttt ccggtttcta aacgtcatttaattgaagag gggcaaggtt 4860 tctccctgct tagctgggga acagtaaatt gacttattccctctggaaga cagggtttaa 4920 gagagccctg acctactcta tttaccaagg tcaaggaatgggatgacctg caatttgtaa 4980 accatcaaac ttctttacac aaaggtaaac aaacaaacaaagcctttgat tacatggccc 5040 atgcctcgaa acttgtgacc taaaaacaac ggaggggggggggcggaggg aatctgtaca 5100 ttttagtaat gatcttgcct tactaaatgt ttaaagtgattcatctatga catatgaacc 5160 attttcttcc ttttgagctg gtaagagcta gagtgtgcacacttgtaagg gaaggaacag 5220 gcctccctgg gcagcaaatg cacacagggc gatatatgggataaaaacaa attatgcaga 5280 aatacaagaa tggatggtgc ccattattgc agcacatgtcttacacccat tttcaacagt 5340 gctgaatacg gaatatgaag aaggcctttc aaatgccccctgcccccccc ctccagcatt 5400 aagccgctct gagtttcact tggcagtgaa gcattaaagatatagtgtac cctggaaata 5460 taaattatta atatattcaa catttttgtc acttctcaactgttaaccaa attagacaaa 5520 actttctttt gctaagtaga ttttctcttt aggattagctttgggagtgt catagggcca 5580 ccgcagtttt aaaaaagaga gagagtcatt tacatataattttgcaaact tccactggtc 5640 catgacagtc tttgtacctc aaatgttggt attcgagatttctttttttt tctttccttc 5700 ttatttaact gtggaggata aaagcctgct ttggatttacaatgacttta ggataaaagt 5760 ctctctctct ctctctctct ctctctctct ctctctctgtgtgtgtgtgt gtgtgtgtgt 5820 gtgtgtgtgt gtgtgttgtc tttttttttt cctgaaaatytcatgcaaaa attccaattc 5880 tttaggcttc tccaaaagtc aactgtggaa tattgcatctttgcaatagg ctgaaaacat 5940 cacttcaaag tggaatgaat tcccattgca tagtgactttagggcgctct atcacacttc 6000 tttttctggt cacatactcc ctcgccccag cttctgcactgcagacccac cccttccaac 6060 tctacccccc ccccccccgt ctgagtttct taggcccaatcctactcttt ctttaagtaa 6120 gttgtatagt aatcgtttcc aagagaggcc caaccaaacccagaaataac agaacacaaa 6180 aaccccgtgg gtttggctgg aggtaagcca ttgtgacggctctgaaaggg ggaccctctt 6240 gttccttggg cttcatcctt gagtgggcaa gaataaatataatcatcccc atgaaatcaa 6300 aggtttttct ccagaggctc gaggaacaaa gctcgtggctggtgctttgg gcggtcccgt 6360 ggccgcgaag gcgccccagc caccctgcag aaagggtggtaaatacagag ctcacttgtg 6420 cttccaagca ttgttgaaca aagacggccc catgtatagtgggggagccc ttctaatctg 6480 ggctgctgga tgccaggaaa gaaaggtgag agaaatgtccccaaagatag ggtcttctct 6540 tgtttgtgtt ttgaatgagt gagatgatga catagatagcaagccaaaat aaaagaataa 6600 atagtaagta agccagtgat agatgataga acacagagatagatagatag atagatagat 6660 agatagatag atagatagat agatggatgg atggacggatgaatggatag atagatagca 6720 tcattaactc aggtactcac agcaactagg gaaacggattgaaaaactct tagaaactgg 6780 taatggatgt ctatacagag tcacacacca agaagactgtgtgtgtggat catccatata 6840 tcaggcacct gatcagtatt tgtggaatgg ccgaaggaattaacagccca gtagagccga 6900 atctgttaga attctgaatc aaaatctctc agatgtctcccttagatgta tccatctagg 6960 catctgtggc atagtctaca cgcacactga aggttatcagagaagggcaa ttaattgctc 7020 tgggtcgttt tttcttttct tacctatgtt ctgacactgggagacaatgc ctgccttgct 7080 tggttaaggt aattagctct tactgtaggc gtaggtaacggcacagatgg caattcggtg 7140 actcaggctc agaactcctg ggttcctgtc cttttgtgtggaacagctac atggctgaac 7200 cgacccagac agacttggcc ctgactccat taggtactcagaggctactg tctgaagcca 7260 gtctctcaat ttacacagta ctatggcttc agtaggatgtacgtctgctg catggtctgg 7320 ggcatggaat acccaacatg catctctcat catgcggctgcaatagcttg aatctgaaat 7380 gtagtccagg gctcatattt tgttcacaca cccagtcctcagccagagtt actgtttttg 7440 ttttgggagg ctgtggaacc tttcggagat gggttcagctgaaggaggtg ggctacaggg 7500 ggcaggtctt cgggatatgg taacctcccc tacttgttatcttgcactct gtttcctggt 7560 ctgccctgag gtgagcagct gcagccacac actcccaaggtcatgactca ggcacctccg 7620 tcctgcccca ccctcctgtc accacgtgcg gagccctctgcaacagtgag ctgagcttaa 7680 tcttactacc cctcttaggt tgcttctgcc ctctactgtggttatagaga cacaaacgta 7740 actaacatga ttgctatcaa gattaagagg attagtatattcaaaatgct ctagacagcg 7800 ctaaagatat agtagacatt caataaatgt tagctgtggccgctggcttt tccacctgtg 7860 ccctcgtacc cgaataaaat ctcagaggct taatattatttatgaatgcc taggctataa 7920 gctaggctgg tttcccaact aattcttttt ttaaaaaaatatgtatttgt tttatgtata 7980 tgaaaattac agatgctgca gaagtctggg gagatccaaagtcactgcag aagtcttagg 8040 aggattaagg tatccagtgt ttatagtggt gggtccctcctgtagctgtc atcagacaca 8100 ccagaagagg gcatcagatc ccattacaga tgcttgtgagtcaccatgtg gatgatagga 8160 attgaactca ggacctctgg aagggctgag ccatctctccagaccacacc ccccgccccg 8220 cccccgaaac cccatccgtt attatccctt ttattctaacctgatttcag ctatgtggct 8280 ggtttcctct ccttgtcttc ttgccttcca ctctgcctagtggttgaaaa ttcccatgct 8340 gactctctca gccagaagtc ccagctggaa gtctcaccttctaatcctgc ctctgtttat 8400 tggccattag atttttattg gcaggtggtt cttcctcacagtacacagaa gattgtccct 8460 acagcaggcc acctattcct gtcttttctc tttctccctctcaaagaatc agaaccctag 8520 ggtgacctca aactaagtaa caaaatgttc agacaaatagtgaagtcttt acacacacac 8580 acacacacac acacacacac acacacccgt gtttctttaacaaacctttt ctttgtttag 8640 ggaaatgggt tagaatttgt aaatctacct ctgaaacagtctttgggcta tgggctatgg 8700 tgtgcacaat ttctaaagga ctaaagttct cctttaggcaagaattggta aaagccacta 8760 gcaaattttg agtactattt ctcatccctt acaaatatttctgctggatg tggtggcaca 8820 cgcctttaat gcactcagag gcagaggcgg gtcaatttctgagctcaagg ccagcctggt 8880 ctacagaaca agttccagga catccagggc tcacagagaagccctatctc aaaaatgttt 8940 ttctccatgt ttcttacata ttttatacat aaaattctacatgaagttgc agatatggtt 9000 cagtggttaa gagcattgac tactcttcca gaggtcctgagttcagttcc cagcaaccac 9060 accgtagctc acaactatct atatgggatc tgatgccctcttctggtgag tctaaaggga 9120 gcaacagtat actgacatat ataaaataaa taaataaatcttttaaaaaa ttctacatga 9180 atacaggata ttgcatggca tctaataaat aatacattgtatagtaagtg ggtcatggta 9240 gttgatatgg ccatcatttt aagcatttat atttttaaaatatgtttcga ttgatgcata 9300 gtgatgtact tatctatgtt gataaatgtg acattttgaatacacctata cagtgcataa 9360 ctgtgaagag cgaatgaagt cctgagcaaa tgtgtattatttgtcggagc acagtcatgt 9420 caaggccaca gtgtctcaga cccacggaaa ggcagggccttcccctggtc cgtgtacaga 9480 tcctgaggca ccattgacct tcagctaaaa ccaagggttgcggtaactat cagctacagc 9540 ttcctctgcc aagacaactg caacctgaga ctgctcccccaacagaggcc tcctatattg 9600 agtctagcta ctggactcag ctataaatac ccaccctacctggaggttta tagccaacag 9660 tagatagaaa acatactgag ttttccaggc tgctggtgtgagcccatcag agcacagagc 9720 ccacctaatc ttggtaggac cagtctctgt aggggaggaatctttaggca gtaaacaccc 9780 agaaggaaaa agctatggtg gacattttgc acacatggtcgccattcaca cacagatcca 9840 gaggagggct ttgctagccc cctgagccct agctgaggaaggctttgcca ctttcgtatg 9900 tttgaggcat tgtggttctt ggcaaggcca tgcccatgtgaagcaatgga catctccaca 9960 ggacttcaca tacttcgtat acctgctaat attaggtatcaatctgccta catccaagca 10020 aatattcttt acaaggcagg gtataacact gtagctcctactgtgctttt aatgaatata 10080 taagtttgat tcagaagtga atcgcttcaa gcccactgtccccatttgtt gaattccatc 10140 ctatcccaaa tcacaaaatg aacttttttt tttttttggtctcttcttaa cacttttatc 10200 tagatct 10207 <210> SEQ ID NO 4 <211>LENGTH: 4109 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 4 ttcactccct cctcctcccc ccgatatttt cgctttctaa aaccataaaaaagagatcaa 60 cttcccaaac ttcacagggc ccttttcagt gacaaatatt gatgcccaaagtgtctgcgc 120 tctcccgccc ccaaacgtta agaaaagacg ccagcgaaag ggagacataaaaagttaaca 180 atgctgtgaa aatatgtttg cagaaaatag acaatcgttg gattaaacgtattcaagtat 240 gaaataatgc ctttttgtgt caaaacttgg gcgatgggcg ggtacaaaagttccctgtgg 300 cagctacttg ctccctttgt gacccgtgcg ctttggcgtc tccacttgggcgcattactc 360 agagccctct aagcgcgatt gtttctccct ttctaatgac atttaccggatcaaaacatg 420 ctgttaattc gatcagaagg cttcaccctc cctgacaaag ccacaataatttctcctgaa 480 gtttgttaaa ttgaccaaaa ttaggcaaat gaataggggt ctgtaggcgccccctctcgc 540 aggtgacgtt gcataatgct tgcctgggcc agctgcattc ccccttttcttctcggccca 600 ctcttctccg tgttgccccc caatcctccc acccgcccct tcacacacaaacacacacac 660 acgcccgcca gtctctcctc ccccaccctc tggcattatt aaaatttagcccaatgaaac 720 tattcatact tttcaatgga cattttcgta taggataata ggctacaaattgagcctctt 780 ccccccgcga agagggagca ggaggggtgg ggagaaaaga aagccagcgacgtggtcagg 840 gagtaggggg gagcgtcgcg tgccaaacag cggcgggagg ggcgaggcgaggcgaggcgg 900 gagcctcgtg tgccagccgc agccccacac ctgccgggat gtccggacaaataaagcggt 960 gtaaacaaaa aggggggaga aggacgtgtc accaagtcgt gtgagaaaagcctgggaaca 1020 aacggggcgc ctccgtctcc aagagctctc ccttgaaccc ggcggaacagcctattaaag 1080 gcttacttaa ttactttaat gactctggac aggctttaaa acgcactcggcgctgggacg 1140 gcgggcttgc tgggatttgt aaacaggcga tcatgtgaga ctcagcggtgggactaaaag 1200 aggcgacact gttttgtgag ggtcctcgcc ccccggtgcc cgcggccgctgcgcccctgc 1260 gctccgcgcc cgcgactgct gcaggcgctt gctcgcccgc cggccccagtttaccgcctc 1320 cttttcccgc ccgagctgtt gccatgcaaa tgttattcct gggccgatcacgtgtccttt 1380 gaagggccac gtggattaac aaagctgatc tgcccccagc tcgcccccctcggctgctaa 1440 tttttttttt ttcttaactt ctttaaaact gatcttgaat gcatacatcctccaaacgca 1500 gctcccctaa tcctatggaa taattcaact cgtgaatgca tctggagccctagatgaccg 1560 ctttataagg cagccctcgg agttgggagg ctgcagtcta cctgggacactcgaggaggc 1620 acacgaggcg gaaaagtgga cgggtgcccc gcgccaccgc ctctcccgagggcgcgtact 1680 gaccaggatg tcagacaagc tcaaggaacg caaagtgagt cggctgagcccaaatggcac 1740 ttgcgccctg gtggtggagg catctgactc accgacccgc cacttgggtggaccgatggc 1800 agggaagtgc ccgcacggga ctttgagtgt ggaggaatct cgagtggtttgggaaggggg 1860 tggggtagag agaggagggt gctcaaccag gtagaagtcc tgcctggaggctggcggcca 1920 cctccccaaa ggcctgggta gggtggctgg aaggggagcg cgccagcgcgggcgtcagaa 1980 ggcagagctc acctggcccg agcgtggcgg gccaggagtc ccttcctagagcgaatctga 2040 gcagtgtgcg cggctggaga ggcggcttgc acccagctgc tagagccgcgcctttgacga 2100 cttcccgggc aaagccatcc taaacataca accctctctt cgcagagaacccccgtttct 2160 cataaagtga tagaaaagcg gaggagggac aggatcaacc gctgcttgaacgagctgggc 2220 aagacagtgc ccatggcctt ggcgaagcag gtaacgttgg cgacccggagccgggtgcca 2280 ggcgttggga ggcctctgcg gccactctgc ggagacgccc gagcccgcggacacagagga 2340 cctcacttgc gggcccctcc caggcggggg gcctgagcgc agggcgaacctcaggaggct 2400 ctggcgcaga tccgcagccg cgtccgctcg ctggtcctct ccagcgccacagtgcccgac 2460 cagcaggcgc tgggccgctg cgaggggccc ttcctccttt tgcagagttccgggaagctg 2520 gagaaggcgg agatcctcga gatgaccgtt cagtacctga gagcactgcactccgctgat 2580 tttccccggg gaagggaaaa aggtgggcac aggttaggga atgggacgcctgggccaggc 2640 gcctgcgcca cccagagacc ctggggctgg gaggggagtg actgagtgttcccgagagct 2700 ctactagagc tccactctcc aggagggcag gggtcctgcg aaggcgcctctccctgctcg 2760 gcgacagcaa ggcggcgagg gagaactggt tcccagggag gctggttccacctccttctc 2820 ccgggtgggg tgggtggggt caggctctca gggaaagagg gcgtttgcgcgtgcctcgcc 2880 agtctctaag actgcgcaga ggagagggcg ggcctcaaat gggaactttggccagaaaat 2940 gtggtgggag gtgccctgca cccgctttgg gctcgcgtgg ggaaggggcactcagggtgt 3000 gtccgtccta cgggctttct cgttcctcca gcagaacttc tagcggagtttgccaactac 3060 ttccactatg gctaccacga gtgcatgaag aacctggtgc attacctcaccacggtggag 3120 cggatggaga ccaaggacac gaagtacgcg cgcatcctcg ccttcttgcagtccaaggcc 3180 cgcctgggcg cggagcccgc ctttccgccg ctgggttcgc tcccggagccggatttctcc 3240 tatcagctgc accctgcggg gcccgaattc gctggtcaca gcccgggcgaggccgctgtg 3300 ttcccgcagg gctctggtgc cgggcctttc ccctggccgc ctggcgcggcccgcagcccc 3360 gcgctgccct acctgcccag cgcgccagtg ccgctcgcta gcccagcgcagcagcacagc 3420 cccttcctga caccggtgca gggcctggac cggcattacc tcaacctgatcggccacgcg 3480 caccccaacg cccttaacct gcacacgccc cagcaccccc cggtgctctgacgcccactc 3540 gcccgccaga tttctcctcg ctttgggcgc ttttaggaga aatgctgtatatattgtaca 3600 cataatgtgt aaatattgta ccccaaaaat ctgggctggg ggaggcaaagagcgaatgag 3660 tcttctgaag gatctccccc tggtaataaa cgttttctga taaagacccaaagagaggga 3720 tttatgtatt accttctgct catccacacc cgtcccctcc gcggtgcccagggcgcaagg 3780 ctgacagggt tcaaggtaac agccctcagt aacttggtga ggggcccgctgtggagtctg 3840 agggaggggg acgttaaatg gaggttcagg cagatattct aggcatcgcttaagggcggg 3900 attcgcggct tcctgcaccc gccccatctt gagcattagc ctcagagaaacaggggaggc 3960 gaagaacact gcccttggct ctcatgaaaa tgcaaattga aggacctgccccaaactgac 4020 ttaggcggac cgttgccagc tgcgacccgg gcgcccgtcc tacccggagccaagtgcccg 4080 acgcgcgcgc accgcacgcc cgcgggtct 4109 <210> SEQ ID NO 5<211> LENGTH: 241 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 5 Met Ser Asp Arg Leu Lys Glu Arg Lys Arg Thr Pro Val Ser HisLys 1 5 10 15 Val Ile Glu Lys Arg Arg Arg Asp Arg Ile Asn Arg Cys LeuAsn Glu 20 25 30 Leu Gly Lys Thr Val Pro Met Ala Leu Ala Lys Gln Ser SerGly Lys 35 40 45 Leu Glu Lys Ala Glu Ile Leu Glu Met Thr Val Gln Tyr LeuArg Ala 50 55 60 Leu His Ser Ala Asp Phe Pro Arg Gly Arg Glu Lys Ala GluLeu Leu 65 70 75 80 Ala Glu Phe Ala Asn Tyr Phe His Tyr Gly Tyr His GluCys Met Lys 85 90 95 Asn Leu Val His Tyr Leu Thr Thr Val Glu Arg Met GluThr Lys Asp 100 105 110 Thr Lys Tyr Ala Arg Ile Leu Ala Phe Leu Gln SerLys Ala Arg Leu 115 120 125 Gly Ala Glu Pro Thr Phe Pro Pro Leu Ser LeuPro Glu Pro Asp Phe 130 135 140 Ser Tyr Gln Leu His Ala Ala Ser Pro GluPhe Pro Gly His Ser Pro 145 150 155 160 Gly Glu Ala Thr Met Phe Pro GlnGly Ala Thr Pro Gly Ser Phe Pro 165 170 175 Trp Pro Pro Gly Ala Ala ArgSer Pro Ala Leu Pro Tyr Leu Ser Ser 180 185 190 Ala Thr Val Pro Leu ProSer Pro Ala Gln Gln His Ser Pro Phe Leu 195 200 205 Ala Pro Met Gln GlyLeu Asp Arg His Tyr Leu Asn Leu Ile Gly His 210 215 220 Gly His Pro AsnGly Leu Asn Leu His Thr Pro Gln His Pro Pro Val 225 230 235 240 Leu<210> SEQ ID NO 6 <211> LENGTH: 241 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 6 Met Ser Asp Lys Leu Lys Glu Arg Lys Arg ThrPro Val Ser His Lys 1 5 10 15 Val Ile Glu Lys Arg Arg Arg Asp Arg IleAsn Arg Cys Leu Asn Glu 20 25 30 Leu Gly Lys Thr Val Pro Met Ala Leu AlaLys Gln Ser Ser Gly Lys 35 40 45 Leu Glu Lys Ala Glu Ile Leu Glu Met ThrVal Gln Tyr Leu Arg Ala 50 55 60 Leu His Ser Ala Asp Phe Pro Arg Gly ArgGlu Lys Glu Leu Leu Ala 65 70 75 80 Glu Phe Ala Asn Tyr Phe His Tyr GlyTyr His Glu Cys Met Lys Asn 85 90 95 Leu Val His Tyr Leu Thr Thr Val GluArg Met Glu Thr Lys Asp Thr 100 105 110 Lys Tyr Ala Arg Ile Leu Ala PheLeu Gln Ser Lys Ala Arg Leu Gly 115 120 125 Ala Glu Pro Ala Phe Pro ProLeu Gly Ser Leu Pro Glu Pro Asp Phe 130 135 140 Ser Tyr Gln Leu His ProAla Gly Pro Glu Phe Ala Gly His Ser Pro 145 150 155 160 Gly Glu Ala AlaVal Phe Pro Gln Gly Ser Gly Ala Gly Pro Phe Pro 165 170 175 Trp Pro ProGly Ala Ala Arg Ser Pro Ala Leu Pro Tyr Leu Pro Ser 180 185 190 Ala ProVal Pro Leu Ala Ser Pro Ala Gln Gln His Ser Pro Phe Leu 195 200 205 ThrPro Val Gln Gly Leu Asp Arg His Tyr Leu Asn Leu Ile Gly His 210 215 220Ala His Pro Asn Ala Leu Asn Leu His Thr Pro Gln His Pro Pro Val 225 230235 240 Leu <210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Primer H2.1<400> SEQUENCE: 7 tcgctgcttg aacgagctg 19 <210> SEQ ID NO 8 <211>LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: Primer H2.10R <400> SEQUENCE: 8 cagagttccgggaaactg 18 <210> SEQ ID NO 9 LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: Primer H2.18D <400>SEQUENCE: 9 gagactggaa ggagagtcc 19 <210> SEQ ID NO 10 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: OTHERINFORMATION: Primer H2.19R <400> SEQUENCE: 10 agggtcacta attcgccaac 20<210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: Primer H2.3 <400>SEQUENCE: 11 tggcaagaca gtccctatgg 20 <210> SEQ ID NO 12 <211> LENGTH:19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: Primer H4R <400> SEQUENCE: 12 ctggttccac ctccttctc 19 <210>SEQ ID NO 13 LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer H5D <400> SEQUENCE: 13ccgctagaag ttctgctgg 19 <210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: OTHER INFORMATION: PrimerMdnPr1D <400> SEQUENCE: 14 ggagccccct cggacct 17 <210> SEQ ID NO 15<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer MdnPr2D <400> SEQUENCE: 15caaacgcaga actcctaatc c 21 <210> SEQ ID NO 16 <211> LENGTH: 108 <212>TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 16 agaaggagagaccgaattaa ccgctgcttg aacgagctgg gcaagacagt ccctatggcc 60 ctggcgaaacagagttccgg gaaactggag aaggcggaga tcctggag 108 <210> SEQ ID NO 17 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: Primer MDN1D <400> SEQUENCE: 17 ctgatcttgaatgcatacat cc 22 <210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer MDN2D <400> SEQUENCE: 18 cccttcctag agcgaatctg ag 22 <210> SEQ IDNO 19 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer MDN3D <400> SEQUENCE: 19gcagggcgaa cctcaggag 19 <210> SEQ ID NO 20 <211> LENGTH: 19 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer MDN4aD <400> SEQUENCE: 20 gtgccctgca cccctttgg 19 <210> SEQ ID NO21 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer MDN4bD <400> SEQUENCE: 21ggcgaggccg ctgtgttcc 19 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer MDN1R <400> SEQUENCE: 22 cgggtcggtg agtcagatgc 20 <210> SEQ ID NO23 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer MDN2R <400> SEQUENCE: 23gggcgtctcc gcagagtgg 19 <210> SEQ ID NO 24 <211> LENGTH: 22 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer MDN3R <400> SEQUENCE: 24 ctcgggaaca ctcagtcact cc 22 <210> SEQ IDNO 25 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer MDN4aR <400> SEQUENCE: 25aggcggccag gggaaagg 18 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer MDN4bR <400> SEQUENCE: 26 ggagatcctt cagaagactc 20 <210> SEQ IDNO 27 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Girella zebra<400> SEQUENCE: 27 Met Arg Arg Asp Arg Ile Asn Lys Cys Ile Glu Gln LeuLys Ile Leu 1 5 10 15 Leu Lys Thr Glu Ile Lys Ala Ser Gln Pro Cys SerLys Leu Glu Lys 20 25 30 Ala Asp Ile Leu Glu Met Ala Val Ile Tyr Leu LysAsn 35 40 45 <210> SEQ ID NO 28 <211> LENGTH: 54 <212> TYPE: PRT <213>ORGANISM: Mus musculus <400> SEQUENCE: 28 His Arg Leu Ile Glu Lys LysArg Arg Asp Arg Ile Asn Glu Cys Ile 1 5 10 15 Ala Gln Leu Lys Asp LeuLeu Pro Glu His Leu Lys Leu Thr Thr Leu 20 25 30 Gly His Leu Glu Lys AlaVal Val Leu Glu Leu Thr Leu Lys His Leu 35 40 45 Lys Ala Leu Thr Ala Leu50 <210> SEQ ID NO 29 <211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 29 His Arg Leu Ile Glu Lys Lys Arg Arg AspArg Ile Asn Glu Cys Ile 1 5 10 15 Ala Gln Leu Lys Asp Leu Leu Pro GluHis Leu Lys Leu Thr Thr Leu 20 25 30 Gly His Leu Glu Lys Ala Val Val LeuGlu Leu Thr Leu Lys His Val 35 40 45 Lys Ala Leu Thr Asn Leu 50 <210>SEQ ID NO 30 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 30 Thr Pro Val Ser His Lys Val Ile Glu Lys ArgArg Arg Asp Arg Ile 1 5 10 15 Asn Arg Cys Leu Asn Glu Leu Gly Lys ThrVal Pro Met Ala Leu Ala 20 25 30 Lys Gln Ser Ser Gly Lys Leu Glu Lys AlaGlu Ile Leu Glu Met Thr 35 40 45 Val Gln Tyr Leu Arg Ala Leu His Ser Ala50 55 <210> SEQ ID NO 31 <211> LENGTH: 58 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 31 Arg Lys Arg Arg Arg Gly IleIle Glu Lys Arg Arg Arg Asp Arg Ile 1 5 10 15 Asn Asn Ser Leu Ser GluLeu Arg Arg Leu Val Pro Ser Ala Phe Glu 20 25 30 Lys Gln Gly Ser Ala LysLeu Glu Lys Ala Glu Ile Leu Gln Met Thr 35 40 45 Val Asp His Leu Lys MetLeu His Thr Ala 50 55 <210> SEQ ID NO 32 <211> LENGTH: 60 <212> TYPE:PRT <213> ORGANISM: Girella zebra <400> SEQUENCE: 32 Arg Arg Val Pro LysPro Leu Met Glu Lys Arg Arg Arg Asp Arg Ile 1 5 10 15 Asn Gln Ser LeuGlu Thr Leu Arg Met Leu Leu Leu Glu Asn Thr Asn 20 25 30 Asn Glu Lys LeuLys Asn Pro Lys Val Glu Lys Ala Glu Ile Leu Glu 35 40 45 Ser Val Val HisPhe Leu Arg Ala Glu Gln Ala Ser 50 55 60 <210> SEQ ID NO 33 <211>LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:33 Asn Arg Leu Arg Lys Pro Val Val Glu Lys Met Arg Arg Asp Arg Ile 1 510 15 Asn Ser Ser Ile Glu Gln Leu Lys Leu Leu Leu Glu Gln Glu Phe Ala 2025 30 Arg His Gln Pro Asn Ser Lys Leu Glu Lys Ala Asp Ile Leu Glu Met 3540 45 Ala Val Ser Tyr Leu Lys His 50 55 <210> SEQ ID NO 34 <211> LENGTH:60 <212> TYPE: PRT <213> ORGANISM: Girella zebra <400> SEQUENCE: 34 LysArg Ile Leu Lys Pro Val Ile Glu Lys Lys Arg Arg Asp Arg Ile 1 5 10 15Asn Gln Arg Leu Glu Glu Leu Arg Thr Leu Leu Leu Asp Asn Thr Leu 20 25 30Asp Ser Arg Leu Gln Asn Pro Lys Leu Glu Lys Ala Glu Ile Leu Glu 35 40 45Leu Ala Val Glu Tyr Ile Arg Thr Lys Thr Ala Thr 50 55 60 <210> SEQ ID NO35 <211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Drosophilamelanogaster <400> SEQUENCE: 35 Arg Lys Thr Asn Lys Pro Ile Met Glu LysArg Arg Arg Ala Arg Ile 1 5 10 15 Asn His Cys Leu Asn Glu Leu Lys SerLeu Ile Leu Glu Ala Met Lys 20 25 30 Lys Asp Pro Ala Arg His Thr Lys LeuGlu Lys Ala Asp Ile Leu Glu 35 40 45 Met Thr Val Lys His Leu Gln Ser ValGln Arg 50 55 <210> SEQ ID NO 36 <211> LENGTH: 59 <212> TYPE: PRT <213>ORGANISM: Drosophila melanogaster <400> SEQUENCE: 36 Arg Arg Ser Asn LysPro Ile Met Glu Lys Arg Arg Arg Ala Arg Ile 1 5 10 15 Asn Asn Cys LeuAsn Glu Leu Lys Thr Leu Ile Leu Asp Ala Thr Lys 20 25 30 Lys Asp Pro AlaArg His Ser Lys Leu Glu Lys Ala Asp Ile Leu Glu 35 40 45 Lys Thr Val LysHis Leu Gln Glu Leu Gln Arg 50 55 <210> SEQ ID NO 37 <211> LENGTH: 58<212> TYPE: PRT <213> ORGANISM: Girella zebra <400> SEQUENCE: 37 Arg LysSer Ser Lys Pro Ile Met Glu Lys Arg Arg Arg Ala Arg Ile 1 5 10 15 AsnGlu Ser Leu Gly Gln Leu Lys Thr Leu Ile Leu Asp Ala Leu Lys 20 25 30 LysAsp Ser Ser Arg His Ser Lys Leu Glu Lys Ala Asp Ile Leu Glu 35 40 45 MetThr Val Lys His Leu Arg Asn Met Gln 50 55 <210> SEQ ID NO 38 <211>LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:38 Arg Lys Ser Ser Lys Pro Ile Met Glu Lys Arg Arg Arg Ala Arg Ile 1 510 15 Asn Glu Ser Leu Ser Gln Leu Lys Thr Leu Ile Leu Asp Ala Leu Lys 2025 30 Lys Asp Ser Ser Arg His Ser Lys Leu Glu Lys Ala Asp Ile Leu Glu 3540 45 Met Thr Val Lys His Leu Arg Asn Leu Gln 50 55 <210> SEQ ID NO 39<211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 39 Arg Lys Ser Ser Lys Pro Val Met Glu Lys Arg Arg Arg Ala ArgIle 1 5 10 15 Asn Glu Ser Leu Ala Gln Leu Lys Thr Leu Ile Leu Asp AlaLeu Arg 20 25 30 Lys Glu Ser Ser Arg His Ser Lys Leu Glu Lys Ala Asp IleLeu Glu 35 40 45 Met Thr Val Arg His Leu Arg Ser Leu Arg 50 55 <210> SEQID NO 40 <211> LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 40 Arg Lys Asn Leu Lys Pro Leu Leu Glu Lys Arg Arg ArgAla Arg Ile 1 5 10 15 Asn Glu Ser Leu Ser Gln Leu Lys Gly Leu Val LeuPro Leu Leu Gly 20 25 30 Ala Glu Thr Ser Arg Ser Ser Lys Leu Glu Lys AlaAsp Ile Leu Glu 35 40 45 Met Thr Val Arg Phe Leu Gln 50 55 <210> SEQ IDNO 41 <211> LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Drosophilamelanogaster <400> SEQUENCE: 41 Arg Lys Val Met Lys Pro Leu Leu Glu ArgLys Arg Arg Ala Arg Ile 1 5 10 15 Asn Lys Cys Leu Asp Glu Leu Lys AspLeu Met Ala Glu Cys Val Ala 20 25 30 Gln Thr Gly Asp Ala Lys Phe Glu LysAla Asp Ile Leu Glu Val Thr 35 40 45 Val Gln His Leu Arg Lys Leu Lys Glu50 55 <210> SEQ ID NO 42 <211> LENGTH: 58 <212> TYPE: PRT <213>ORGANISM: Girella zebra <400> SEQUENCE: 42 Lys Lys Val Ser Lys Pro LeuMet Glu Lys Lys Arg Arg Ala Arg Ile 1 5 10 15 Asn Lys Cys Leu Asn GlnLeu Lys Ser Leu Leu Glu Ser Ala Cys Ser 20 25 30 Asn Asn Ile Arg Lys ArgLys Leu Glu Lys Ala Asp Ile Leu Glu Leu 35 40 45 Thr Val Lys His Leu ArgHis Leu Gln Asn 50 55 <210> SEQ ID NO 43 <211> LENGTH: 51 <212> TYPE:PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 43 Met Glu Lys Lys ArgArg Ala Arg Ile Asn Val Ser Leu Glu Gln Leu 1 5 10 15 Arg Ser Leu LeuGlu Arg His Tyr Ser His Gln Ile Arg Lys Arg Lys 20 25 30 Leu Glu Lys AlaAsp Ile Leu Glu Leu Ser Val Lys Tyr Met Arg Ser 35 40 45 Leu Gln Asn 50<210> SEQ ID NO 44 <211> LENGTH: 63 <212> TYPE: PRT <213> ORGANISM:Girella zebra <400> SEQUENCE: 44 Arg Lys Leu Leu Lys Pro Gln Val Glu ArgArg Arg Arg Glu Arg Met 1 5 10 15 Asn Arg Ser Leu Glu Asn Leu Lys LeuLeu Leu Leu Gln Gly Pro Glu 20 25 30 His Asn Gln Pro Asn Gln Arg Arg LeuGlu Lys Ala Glu Ile Leu Glu 35 40 45 Tyr Thr Val Leu Phe Leu Gln Lys AlaAsn Glu Ala Ser Lys Glu 50 55 60 <210> SEQ ID NO 45 <211> LENGTH: 55<212> TYPE: PRT <213> ORGANISM: Girella zebra <400> SEQUENCE: 45 Asn LysLeu Arg Lys Pro Met Val Glu Lys Ile Arg Arg Glu Arg Ile 1 5 10 15 AsnSer Ser Ile Glu Lys Leu Lys Thr Leu Leu Ala Gln Glu Phe Ile 20 25 30 LysGln Gln Pro Asp Ser Arg Gln Glu Lys Ala Asp Ile Leu Glu Met 35 40 45 ThrLeu Asp Phe Leu Arg Arg 50 55 <210> SEQ ID NO 46 <211> LENGTH: 57 <212>TYPE: PRT <213> ORGANISM: Girella zebra <400> SEQUENCE: 46 Arg Lys LeuArg Lys Pro Leu Ile Glu Lys Lys Arg Arg Glu Arg Ile 1 5 10 15 Asn SerSer Leu Glu Gln Leu Lys Gly Ile Met Val Asp Ala Tyr Asn 20 25 30 Leu AspGln Ser Lys Leu Glu Lys Ala Asp Val Leu Glu Ile Thr Val 35 40 45 Gln HisMet Glu Asn Leu Gln Arg Gly 50 55 <210> SEQ ID NO 47 <211> LENGTH: 42<212> TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: Consensus sequence <400> SEQUENCE: 47 Arg Lys Lys Pro LeuMet Glu Lys Arg Arg Arg Asp Arg Ile Asn Ser 1 5 10 15 Leu Gln Leu LysLeu Leu Leu Asp Ala Leu Leu Glu Lys Ala Asp Ile 20 25 30 Leu Glu Met ThrVal Lys His Leu Arg Leu 35 40

What is claimed is:
 1. A purified and isolated DNA encoding a mammalianbHLH transcription factor for the induction of neural cells.
 2. The DNAof claim 1, encoding a mouse bHLH transcription factor.
 3. The DNA ofclaim 1, which is a cDNA comprising the sequence shown in Seq. ID. No. 1or a portion thereof, which encodes a biologically active transcriptionfactor.
 4. The DNA of claim 1, which comprises the nucleotide sequencefrom nucleotide 145 through 252 of Seq. ID. No.
 1. 5. The DNA of claim1, which is a genomic DNA comprising the sequence shown in Seq. ID. No.3 or a portion thereof, which encodes a biologically activetranscription factor.
 6. The DNA of claim 1, encoding a human bHLHtranscription factor.
 7. The DNA of claim 1, which is a cDNA sequencecomprising the sequence shown in Seq. ID. No. 2 or a portion thereof,which encodes a biologically active transcription factor.
 8. The DNA ofclaim 1, which is a genomic DNA comprising the sequence shown in Seq.ID. No. 4 or a portion thereof, which encodes a biologically activetranscription factor.
 9. An isolated nucleotide which comprises thecomplement of any one of the nucleotides of claim
 1. 10. Purifiedisolated mammalian transcription factor for the induction of neuralcells encoded by the DNA of claim 1 or variants thereof, provided thatsaid variants comprise nucleic acid changes due to the degeneracy of thegenetic code, which code for the same or functionally equivalenttranscription factor as the nucleic acid of claim 1 or provided thatsaid variants hybridize under stringent conditions to a nucleic acidwhich comprises the sequence of claim 1 and further provided that saidvariants code for a protein with activity as transcription factor forthe induction of neural cells.
 11. Purified isolated mouse transcriptionfactor for the induction of neural cells comprising the amino acid ofSeq. ID. No. 5 and homologues or fragments thereof which retainbiological activity.
 12. Purified isolated human transcription factorfor the induction of neural cells comprising the amino acid of Seq. ID.No. 6 and homologues or fragments thereof which retain biologicalactivity.
 13. A fusion protein, comprising the transcription factor ofclaim 10 fused to a signal peptide, which allows the delivery of saidtranscription factor into a target cell.
 14. The fusion protein of claim13, wherein the signal peptide is the HIV-1 TAT sequence and optionallyfurther comprises a His-tag and/or an epitope tag.
 15. An expressionvector comprising the DNA of claim 1 or a DNA, which codes for thefusion protein of claim 13 or
 14. 16. A host cell transformed with thevector of claim
 15. 17. The host cell of claim 16, which is a vertebratestem cell.
 18. The host cell of claim 17, which is a mammalian stemcell.
 19. The host cell of claim 18, which is a mouse stem cell.
 20. Thehost cell of claim 19, which is a human stem cell.
 21. The host cell ofclaim 20, which is an embryonic stem cell.
 22. The host cell of claim20, which is an adult stem cell.
 23. A method for producing thetranscription factor of claim 10 in a substantially pure form, whichcomprises transforming a host cell of claim 16 with the vector of claim15, culturing the host cell under conditions which permit expression ofthe sequence by the host cell and isolating the peptide from the hostcell.
 24. An antibody which specifically binds to the protein of claim10.
 25. The antibody of claim 24, wherein said antibody is selected fromthe the group consisting of a polyclonal antibody, a monoclonalantibody, a humanized antibody, a chimeric antibody, and a syntheticantibody.
 26. The antibody of claims 24 and 25 wherein said antibodiesare linked to a chemotherapeutic agent or toxic agent and/or to animaging agent.
 27. A hybridoma which produces a monoclonal antibodyhaving binding specificity to any one of the proteins of claim
 10. 28. Arecombinant non-human vertebrate in which the DNA of claim 1 has beeninactivated.
 29. A recombinant mouse, in which the DNA of claim 5 hasbeen inactivated.
 30. A nucleic acid probe comprising a nucleic acidsequence complementary to any one of the nucleic acid sequences of claim1 or a portion thereof.
 31. A test kit, comprising the probes of claim30 and means for detecting or measuring the hybridization of said probesto the sequences comprised of claim
 1. 32. An ex vivo method ofproducing dopaminergic neurons, which comprises the following steps: a)providing neural embryonic stem cells, neural adult stem cells and/orembryonic stem cells; b) contacting said cells with an effective amountof the transcription factor of claim 10; c) culturing said cells underconditions, which allow the specification and differentiation todopaminergic neurons; and d) recovering the dopaminergic neurons. 33.Dopaminergic neurons, which are obtainable by the method of claim 32.34. A composition, which comprises an effective amount of thedopaminergic neurons of claim 33 in combination with a pharmaceuticallyacceptable carrier.
 35. A composition comprising an effective amount ofa protein of claim 10, in combination with a pharmaceutically acceptablecarrier.
 36. A composition comprising nucleic acid sequences of claim 1.37. A composition comprising a host cell of claim
 16. 38. A compositioncomprising the antibody of claim
 24. 39. Use of the composition of claim38 in the in vitro or in vivo diagnosis of neurodegenerative disorders.40. A method of treating a patient suffering from a neurodegenerativedisease comprising administering an effective amount of the compositionof claim 34, 35 or 36 to said patient, thereby substituting degeneratedor lost nerval cells in said patient.
 41. The method of claim 40,wherein the compositions are administered intracerebrally.
 42. Themethod of claim 40, wherein the compositions are administeredintraperitoneally.
 43. The method of claim 40, wherein theneurodegenerative disease is Parkinson's disease.
 44. The method ofclaim 40, wherein a vertebrate is treated.
 45. The method of claim 40,wherein a mammal, preferably a human patient is treated.